Dominant negative mutant krp protein protection of active cyclin-cdk complex inhibition by wild-type krp

ABSTRACT

Disclosed are mutant CDK inhibitor (CKI) polypeptides having dominant negative antagonist activity against wild-type CKI proteins, as well as related compositions, including nucleic acids and vectors encoding the mutant CKI polypeptides and transformed host cells and transgenic plants comprising such nucleic acids and vectors. Also disclosed are related methods for using the mutant proteins to modulate cell division in cells, particularly plant cells.

RELATED APPLICATIONS

This application claims priority to U.S. Application No. 60/703,999,filed Jul. 29, 2005, incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Plants have the same basic cell cycle as eukaryotes. Not surprising,they also have in common with eukaryotes kinases called cyclin dependentkinases (CDK) that regulate the transitions between different phases ofthe cell cycle. Arabidopsis, a model plant system used to study the cellcycle have several CDK sub-groups. CDKA is most similar to the cdc2(CDK1) of mammals and contains the highly conserved Pro Ser Thr Ala IleArg Glu (PSTAIRE) amino acid sequence (SEQ ID NO:1) in a region thatmediates the interaction with their cyclin partner. Arabidopsis also hasa plant-specific group of CDK called CDKB that is not conserved inhigher animals. Plants do however lack the mammalian counterpart to theG₁ CDKs-CDK4 and CDK6. CDKA has been proposed to be the G₁ CDK (that isactivated by the plant D-type cyclins), while CDKB has been demonstratedto be predominantly expressed in S-phase and later and, therefore,likely identified as the G₂/M specific CDK.

Activation/inactivation of these CDKs drives cells through the cellcycle and also dictates when cells are to exit the cell cycle.Arabidopsis contains up to 49 cyclins grouped into 10 subclasses (seeWang et al., Plant Physiol. 135:1084-1099, 2004). Only the A, B andD-classes appear to play a role in the cell cycle and activate CDKs(Wang et al., Plant Physiol. 135:1084-1099, 2004). CDKA is activated bythe D-type cyclins, while CDKB is activated by A- and B-type cyclins.

In animals CDKs are negatively regulated by two families of CDKinhibitors (CKIs). One class, called inhibitor of CDK4 (INK4) iscomprised of 4 members (p15, p16, p18 and p19) that bind to and inhibitthe G₁ CDKs, namely CDK4 and CDK6, from binding the cyclin. The othergroup of inhibitors is called Kinase Inhibitor Proteins (KIPs) or CIP(CDK Interacting Protein) proteins and they are highly conserved in allanimals. The CIP/KIP family predominantly inhibit the cyclin A- andE-CDK2 kinase activity. In plants, putative CKIs have been identified(Wang et al., Nature 386:451-452, 1997; Wang et al., Plant J.15:501-510, 1998; De Veylder et al., Plant Cell 13:1653-1667, 2001;Jasinski et al., Plant Physiol. 130:1871-1882, 2002) and shown toinhibit purified cyclin/CDK kinase activity in vitro (Wang et al, 1997,supra; Wang et al., Plant J. 24:613-623, 2000; Lui et al., Plant J.21:379-385, 2000). Expression of plant CKIs showed reduced growth withsmaller organs containing larger cells (see Wang et al., 2000, supra;Jasinski et al., J. Cell Sci. 115:973-982, 2001; De Veylder et al.,supra; Zhou et al., Plant Cell Rep. 20:967-975, 2002; Zhou et al., PlantJ. 35:476-489, 2003; Schnittger et al., Plant Cell 15:303-315, 2003). InArabidopsis, these CKIs, are called Inhibitors of CDK (ICKs) or KIPrelated proteins (KRPs). Seven ICK family members have been identifiedthat most closely resemble the CIP/KIP family of CKIs. Each of theseICK/KRP family members has high amino acid sequence identity top27^(KIP1) but the identity is limited to the most C-terminal 30 aminoacids. To date, no INK related CKIs have been identified in any plant.

Over expression of cyclins or knockouts of CKI illustrate that the wellbalanced cell cycle engine can easily be perturbed in mammals. Thisimbalance can ultimately lead to accelerated cell cycles, increasedanimal size and/or tumor development. Reducing or completely eliminatingCKI “activity” results in increased cyclin/CDK kinase activity. Thisincreased activity results in phosphorylation of downstream targetsnecessary for cell cycle progression and animals ultimately yields cellhyper-proliferation (Coats et al., Science 272:877-880, 1996). Deletionof the p27^(KIP1) gene in mice results in larger mice due to excessivecyclin/cdk activity that leads to excessive cell proliferation (Fero etal., Cell 85:733-744, 1996; Kiyokawa et al., Cell 85:721-732, 1996;Nakayama et al., Cell 85:707-720, 1996).

Mechanisms exist to suppress expression of various members of the KRPfamily. Post-transcriptional gene silencing (PTGS) in plants is anRNA-degradation mechanism similar to RNA interference (RNAi) in animals.RNAi results in the specific degradation of double-stranded RNA (dsRNA)into short 21-23 bp dsRNA fragments which ultimately play a role in thedegradation of a population of homologous RNAs. In plants, PTGS uses aninverted repeats (IR) strategy to suppress gene expression in manyplants species including crop plants such as corn, soy and Canola toname a few. However, IR technology has several drawbacks such as theefficiency of IR sequence, off target gene regulation (Jackson et al.,Nature Biotech. 21:635-637, 2003), transient silencing, overall IRstability, and the like. These drawbacks are compounded in the presentcase by the need to silence more than one gene at a time.

Conventional plant breeding has been the principle driving force forincreased crop yields over the past 75 years (J. Femandez-Cornejo,Agriculture Information Bulletin No. (AIB786) 81 pp, February 2004).More recently, transgenic crops have become available that for examplehave resistance to insect pests and herbicides. However, thesetransgenic crops do come with a yield penalty (Elmore et al., Agron. J93:408-412, 2001; Elmore et al., Agron. J. 93:404-407, 2001). To date,no known transgenic crop is commercially available that has an increasein seed size or an increase in crop yield.

There is a need in the art for improved methods of modifyingcharacteristics of certain commercially valuabile crops, including forexample, but not limitation, increasing crop yields, increasing seedsize, increasing the rate of germination, increasing root mass, and thelike. The present invention as described herein meets these and otherneeds.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a variant plant cyclin dependent kinaseinhibitor (CKI) polypeptide having at least one modification relative toa wild-type CKI polypeptide. In certain embodiments, the modification(s)are within the CDK binding region so as to confer, relative to thewild-type CKI protein, modified binding affinity for CDK protein, whilesubstantially maintaining binding affinity for a cyclin protein. Thevariant CKI polypeptides of the present invention have dominant negativeantagonist activity against wild-type CKI function. When the variant isexpressed within a cell expressing the corresponding wild-type CKIprotein, or a cell expressing a wild-type CKI heterologous to thecorresponding wild-type protein but having substantially equivalentwild-type function with respect to, inter alia, cyclin and CDK binding,the wild-type CKI biological activity is inhibited, leading toaccelerated progression through the cell cycle and increased cellproliferation.

In other aspects, the present invention provides a recombinant nucleicacid encoding a variant CKI polypeptide, or a vector comprising therecombinant nucleic acid. The variant CKI-encoding nucleic acid orvector can be introduced into a host cell for amplification orexpression of the nucleic acid. The host cells can be used, for example,in methods of the invention for producing the variant CKI polypeptides.Further, expression of the variant CKI polypeptide in a cell can be usedin methods of the invention for modulating cell division. For example,expression of variant CKI polypeptide in a cell can lead to acceleratedprogression through the cell cycle and increased cell proliferation.

In still other aspects, a transgenic plant comprising a transgeneencoding the variant CKI polypeptide is provided. Expression of thevariant CKI polypeptide in transgenic plants can be used in methods ofthe invention for, e.g., increasing plant vigor, increasing root mass,increasing plant size, or increasing early germination, and the like.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar to those described herein can be used in the practiceor testing of the present invention, only exemplary methods andmaterials are described. For purposes of the present invention, thefollowing terms are defined below.

The terms “a,” “an,” and “the” as used herein include plural referents,unless the context clearly indicates otherwise.

As used herein, the term “cyclin dependent kinase inhibitor” (alsoreferred to herein as “CDK inhibitor” or “CKI”) refers to a class ofproteins that negatively regulate cyclin dependent kinases (CDKs). CKIsamenable to the present invention are those having separate polypeptideregions capable of independently binding a cyclin and a CDK. Such CKIsinclude, for example, identified families of plant CKIs (the sevenidentified Arabidopsis CKIs), having homology to Kinase InhibitorProteins (KIPs) in animals, referred to as KIP-related proteins (KRPs)(also known as Inhibitors of “CDKs,” or “ICKs”).

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions). Specifically, degenerate codonsubstitutions can be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (see, e.g., Batzer et al.,Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem.260:2605-2608, 1985; Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).The term nucleic acid is used interchangeably with gene, cDNA, and mRNAencoded by a gene.

The term “naturally occurring,” in the context of CKI polypeptides andnucleic acids, means a polypeptide or nucleic acid having an amino acidor nucleotide sequence that is found in nature, i.e., an amino acid ornucleotide sequence that can be isolated from a source in nature (anorganism) and which has not been intentionally modified by humanintervention. As used herein, laboratory strains of plants which mayhave been selectively bred according to classical genetics areconsidered naturally-occurring plants.

As used herein, “wild-type CKI gene” or “wild-type CKI nucleic acid”refers to a sequence of nucleic acid, corresponding to a CKI geneticlocus in the genome of an organism, that encodes a gene productperforming the normal function of the CKI protein encoded by anaturally-occurring nucleotide sequence corresponding to the geneticlocus. A genetic locus can have more than one sequence or allele in apopulation of individuals, and the term “wild-type” encompasses all suchnaturally-occurring alleles that encode a gene product performing thenormal function. “Wild-type” also encompasses gene sequences that arenot necessarily naturally occurring, but that still encode a geneproduct with normal function (e.g., genes having silent mutations orencoding proteins with conservative substitutions).

The term “wild-type CKI polypeptide” or “wild-type CKI protein” refersto a CKI polypeptide encoded by a wild-type gene. A genetic locus canhave more than one sequence or allele in a population of individuals,and the term “wild-type” encompasses all such naturally-occurringalleles that encode a gene product performing the normal function.

The term “mutant” or “variant,” in the context of CKI polypeptides andnucleic acids of the present invention, means a polypeptide or nucleicacid that is modified relative to a corresponding wild-type polypeptideor nucleic acid.

The term “reference CKI polypeptide” is a term used herein for purposesof defining a mutant or variant CKI polypeptide: the term refers to aCKI polypeptide to which a mutant CKI polypeptide is compared forpurposes of defining modifications in amino acid sequence. Thus, amutant CKI polypeptide “comprising a CKI amino acid sequence having atleast one modification relative to a reference CKI polypeptide” meansthat, except for the one or more amino acid modification(s), the mutantCKI polypeptide otherwise comprises the amino acid sequence of thereference polypeptide. In carrying out the present invention asdescribed herein, reference CKI polypeptides are predetermined. Areference CKI polypeptide can be, for example, a wild-type and/or anaturally occurring CKI polypeptide, or a CKI polypeptide that has beenintentionally modified.

The term “modified binding” or “altered binding” refers to a mutant CKIpolypeptide encoded by a wild-type gene whose reference CKI polypeptidebinds a cyclin/CDK complex. By the term “modified binding” or “alteredbinding” herein refers to the binding of the mutant CKI to thecyclin/CDK kinase complex. The term “modified binding” or “alteredbinding” refers to the relative binding of the mutant CKI polypeptidecompared to the reference CKI polypeptide. “Modified binding” or“altered binding” can refer to a mutant CKI polypeptide that has equalbinding, reduced binding, or equivalent binding to the cyclin/CDKcomplex compared to the reference CKI polypeptide.

“Recombinant” as used herein refers to an amino acid sequence or anucleotide sequence that has been intentionally modified by recombinantmethods. By the term “recombinant nucleic acid” herein is meant anucleic acid, originally formed in vitro, in general, by themanipulation of a nucleic acid by endonucleases, in a form not normallyfound in nature. Thus an isolated mutant or variant CKI nucleic acid, ina linear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. A “recombinant protein”is a protein made using recombinant techniques, i.e., through theexpression of a recombinant nucleic acid as depicted above. Arecombinant protein is distinguished from naturally occurring protein byat least one or more characteristics.

With respect to amino acids, a “non-conservative” modification means amodification in which the wild type residue and the mutant residuediffer significantly in one or more physical properties, includinghydrophobicity, charge, size, and shape. For example, modifications froma polar residue to a nonpolar residue or vice-versa, modifications frompositively charged residues to negatively charged residues or viceversa, and modifications from large residues to small residues or viceversa are nonconservative modifications. For example, substitutions maybe made which more significantly affect: the structure of thepolypeptide backbone in the area of the alteration, for example thealpha-helical or beta-sheet structure; the charge or hydrophobicity ofthe molecule at the target site; or the bulk of the side chain. Thesubstitutions which in general are expected to produce the greatestchanges in the polypeptide's properties are those in which (a) ahydrophilic residue, e.g., seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl oralanyl; (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having a side chain, e.g., glycine.

Conservative modifications are generally those shown below, however, asis known in the art, other substitutions may be considered conservative.

Ala: Ser

Arg: Lys

Asn: Gln, His

Asp: Glu

Cys: Ser

Gln: Asn

Glu: Asp

Gly: Pro

His: Asn, Gln

Ile: Leu, Val

Leu, Ile, Val

Lys: Arg, Gln, Glu

Met: Leu, Ile

Phe: Met, Leu, Tyr

Ser: Thr

Thr: Ser

Trp: Tyr

Tyr: Trp, Phe

Val: Ile, Leu

The term “dominant negative,” in the context of protein mechanism ofaction or gene phenotype, refers to a mutant or variant protein, or thegene encoding the mutant or variant protein, that substantially preventsa corresponding protein having wild-type function from performing thewild-type function.

The phrase “antagonist of a wild-type CKI” as used herein means that amutant CKI polypeptide significantly decreases (inhibits) the ability ofa wild-type CKI polypeptide to inhibit kinase activity of CDK/cyclincomplexes as compared to kinase activity inhibition by the wild-type CKIpolypeptide in the absence of the antagonist.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, a promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation.

The term “plant” includes whole plants, plant organs (e.g., leaves,stems, flowers, roots, and the like), seeds, and plant cells (includingtissue culture cells) and progeny thereof. The class of plants which canbe used in the methods of the invention is generally as broad as theclass of higher plants amenable to transformation techniques, includinggymnosperms and angiosperms, both monocotyledonous and dicotyledonousplants, as well as certain lower plants such as algae. It includesplants of a variety of ploidy levels, including polyploid, diploid andhaploid. Examples of monocotyledonous angiosperms include, e.g.,asparagus, field and sweetcorn, barley, wheat, rice, sorghum, sugarcane, onion, millet, rye and oats and other cereal grains. Examples ofdicotyledonous angiosperms include, but are not limited to, tomato,tobacco, cotton, rapeseed (Canola), camelina, field beans, soybeans,peppers, lettuce, and the like. Examples of woody species includepoplar, pine, cedar, oak, fir, and the like.

A “heterologous sequence” is one that originates from a differentspecies, or, if from the same species, is substantially modified fromits original form. For example, a heterologous promoter operably linkedto a structural gene is from a species different from that from whichthe structural gene was derived, or, if from the same species, issubstantially modified from its original form.

The term “vector” refers to a piece of DNA, typically double-stranded,which may have inserted into it a piece of foreign DNA. The vector orreplicon may be for example, of plasmid or viral origin. Vectors contain“replicon” polynucleotide sequences that facilitate the autonomousreplication of the vector in a host cell. The term “replicon” in thecontext of this disclosure also includes polynucleotide sequence regionsthat target or otherwise facilitate the recombination of vectorsequences into a host chromosome. In addition, while the foreign DNA maybe inserted initially into, for example, a DNA virus vector,transformation of the viral vector DNA into a host cell may result inconversion of the viral DNA into a viral RNA vector molecule. ForeignDNA is defined as heterologous DNA, which is DNA not naturally found inthe host cell, which, for example, replicates the vector molecule,encodes a selectable or screenable marker or transgene. The vector isused to transport the foreign or heterologous DNA into a suitable hostcell. Once in the host cell, the vector can replicate independently ofor coincidental with the host chromosomal DNA, and several copies of thevector and its inserted DNA can be generated. Alternatively, the vectorcan target insertion of the foreign or heterologous DNA into a hostchromosome. In addition, the vector can also contain the necessaryelements that permit transcription of the inserted DNA into a mRNAmolecule or otherwise cause replication of the inserted DNA intomultiple copies of RNA. Some expression vectors additionally containsequence elements adjacent to the inserted DNA that allow translation ofthe mRNA into a protein molecule. Many molecules of mRNA and polypeptideencoded by the inserted DNA can thus be rapidly synthesized.

The term “transgene vector” refers to a vector that contains an insertedsegment of DNA, the “transgene,” that is transcribed into mRNA orreplicated as a RNA within a host cell. The term “transgene” refers notonly to that portion of inserted DNA that is converted into RNA, butalso those portions of the vector that are necessary for thetranscription or replication of the RNA. In addition, a transgene neednot necessarily comprise a polynucleotide sequence that contains an openreading frame capable of producing a protein.

The terms “transformed host cell,” “transformed,” and “transformation”refer to the introduction of DNA into a cell. The cell is termed a “hostcell,” and it may be a prokaryotic or a eukaryotic cell. Typicalprokaryotic host cells include various strains of E. coli. Typicaleukaryotic host cells are plant cells (e.g., canola, soy, rice, or maizecells and the like), yeast cells, insect cells, or animal cells. Theintroduced DNA is usually in the form of a vector containing an insertedpiece of DNA. The introduced DNA sequence may be from the same speciesas the host cell or from a different species from the host cell, or itmay be a hybrid DNA sequence, containing some foreign DNA and some DNAderived from the host species.

In the context of CKI polypeptides and nucleic acids, “correspondence”to another sequence (e.g., regions, fragments, nucleotide or amino acidpositions, or the like) is based on the convention of numberingaccording to nucleotide or amino acid position number, and then aligningthe sequences in a manner that maximizes the number of nucleotides oramino acids that match at each position, i.e., in a manner thatmaximizes the percentage of sequence identity. Because not all positionswith a given “corresponding region” need be identical, non-matchingpositions within a corresponding region may be regarded as“corresponding positions.” Accordingly, as used herein, referral to an“amino acid position corresponding to amino acid position X” of aspecified CKI polypeptide represents referral to a collection ofequivalent positions in other recognized CKI polypeptides and structuralhomologues and families.

In a typical embodiment of the present invention relating to the KRPsuperfamily of CKI polypeptides and nucleic acids, “correspondence” ofamino acid or nucleotide positions are typically determined with respectto amino acids within the CDK binding region, or nucleotides encodingthe CDK binding region. Generally, as compared to other regions of KRPpolypeptides, CDK binding regions of KRP CKIs share substantial sequenceidentity or similarity. Thus, one suitable technique for determining theCDK binding region of a KRP CKI polypeptide is to identify an amino acidregion sharing substantial sequence identity or similarity to a knownCDK binding region of a second KRP CKI (for example, from about aminoacid positions 145-168 of Brassica napus Krp1 (Bn Krp1)). (See, e.g.,FIG. 1, showing an amino acid sequence alignment of several CKI familymembers with BnKrp1.) Once a sequence corresponding to a CDK bindingregion has been determined by sequence alignment, corresponding aminoacid or nucleotide positions can be determined accordingly.

As used herein, “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the sequence in the comparison window cancomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same(e.g., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95%identity over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or a designated region, asmeasured using one of the following sequence comparison algorithms, orby manual alignment and visual inspection. Sequences are “substantiallyidentical” to each other if they are at least about 25%, at least 30%,at least 35%, at least 40%, at least 45%, at least 50%, or at least 55%identical. These definitions also refer to the complement of a testsequence. Optionally, the identity exists over a polypeptide region thatis at least about 6 amino acids residues in length, at least about 15amino acid residues in length, at least about 25 amino acid residues inlength, at least about 35 amino acid residues in length, at least about50 amino acid residues in length, or at least about 100 or more aminoacids in length, or over a nucleic acid region encoding such apolypeptide region. In certain preferred aspects of the presentinvention, a designated region for comparison is a CDK binding region ofa CKI polypeptide, a polypeptide region comprising a portion of such aCDK binding region, or a polypeptide region comprising such a CDKbinding region.

The terms “similarity” or “percent similarity,” in the context of two ormore polypeptide sequences, refer to two or more sequences orsubsequences that have a specified percentage of amino acid residuesthat are either the same or similar as defined by a conservative aminoacid substitution (e.g., 60% similarity, optionally 65%, 70%, 75%, 80%,85%, 90%, or 95% similar over a specified region), when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection.Sequences are “substantially similar” to each other if they are at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, or at least 55% similar to each other. Optionally,this similarity exists over a region that is at least about 6 amino acidresidues in length, at least about 15 amino acid residues in length, atleast about 25 amino acid residues in length, at least about 35 aminoacid residues in length, at least about 50 amino acid residues inlength, or at least about 100 or more amino acids in length.

In certain aspects of the present invention, for determination ofsequence identity or similarity, a designated region for comparison isthe CDK binding region of a CKI polypeptide, a polypeptide regioncomprising a portion of such a CDK binding region, or a polypeptideregion comprising such a CDK binding region.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities or similarities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof contiguous amino acid or nucleotide positions in which a sequence maybe compared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. With respect tocomparison of CKI polypeptides in accordance with the present invention,a comparison window is typically from about 6 to about 200 or morecontiguous amino acids, typically about 6 to about 50, about 6 to about25, about 15 to about 100, about 15 to about 50, about 15 to about 30,about 20 to about 50, or about 25 to about 50 contiguous amino acids.Methods of alignment of sequences for comparison are well known in theart. Optimal alignment of sequences for comparison can be conducted, forexample, by the local homology algorithm of Smith and Waterman (Adv.Appl. Math. 2:482, 1970), by the homology alignment algorithm ofNeedleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search forsimilarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA85:2444, 1988), by computerized implementations of these algorithms(e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Ausubelet al., Current Protocols in Molecular Biology (1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (J. Mol. Evol.35:351-360, 1987). The method used is similar to the method described byHiggins and Sharp (CABIOS 5:151-153, 1989). The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package (e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395, 1984).

Another example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (Nuc. Acids Res.25:3389-3402, 1977), and Altschul et al. (J. Mol. Biol. 215:403-410,1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5887, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, typically less thanabout 0.01, and more typically less than about 0.001.

Gene homologs and orthologs can also be identified using the HomoloGeneresource of the National Center for Biotechnology Information (NCBI). Ahomolog or ortholog of a first gene can encode a gene product that hasthe same or a similar function as the gene product encoded by the firstgene. Another indication that two nucleic acid sequences or polypeptidesare orthologs is that the heterologous gene can complement (e.g.,rescue) a null allele of the endogenous gene in a eukaryotic cellexpression system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an amino acid sequence alignment of Arabidopsis KRP familymembers with Brassica Krp family members. The sequences of theArabidopsis KRPs were obtained from the public database: AtKrp1 (Genbank# U94772) (SEQ ID NO:2); AtKrp2 (Genbank#CAB76424) (SEQ ID NO:3); AtKrp3(Genbank # CAC41617) (SEQ ID NO:4); AtKrp4 (Genbank # CAC41618) (SEQ IDNO:5); AtKrp5 (Genbank # CAC41619) (SEQ ID NO:6); AtKrp6 (Genbank #CAC41620) (SEQ ID NO:7); and AtKrp7 (Genbank # CAC41621) (SEQ ID NO:8).The sequences for the Brassica KRPs (BnKrp1 (SEQ ID NO:68), BnKrp3 (SEQID NO:69), BnKrp4 (SEQ ID NO:70), BnKrp5 (SEQ ID NO:71), and BnKrp6 (SEQID NO:72)) were obtained as described in Example 1, infra. Also shown isthe amino acid sequence for p27 (SEQ ID NO:73).

FIG. 2 shows an amino acid sequence alignment of corn (SEQ ID NO:9),canola, soy (SEQ ID NO:11), poplar (SEQ ID NO:12), tobacco (SEQ IDNO:13), wheat, rice (SEQ ID NO:15), and potato (SEQ ID NO:16) cyclin andCDK binding domains, illustrating the high degree of sequence identitywithin the CDK binding domain.

FIG. 3 shows mutant BnKrp1 nucleic acid sequences that have been codonoptimized for expression in Maize, coding for either BnKrp1 F151A; 153A(SEQ ID NO:74) or BnKrp1 Y149A; F151A; F153A (SEQ ID NO:75).

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises compositions and methods for modulatingplant cell division. In particular, provided are polypeptides thatantagonize wild-type CKI protein function via a dominant negativemechanism, as well as related polynucleotides, host cells, transgenicplants, and methods for use thereof. A wide variety of transgenicvectors, containing a polynucleotide encoding a variant, dominantnegative CKI polypeptide, can be used to practice the present invention.When the CKI transgenes of the present invention are introduced intoplants and expressed, plant cell division is modulated. The modulationof cell division may occur throughout the plant or in a tissue or organspecific manner depending upon the type of promoter sequence operablylinked to the variant CKI transgene. In particular, the compositions andmethods provided herein can be used to accelerate progression of plantcells through the cell cycle, with a concomitant increase in cellularproliferation. Using the compositions and methods in this manner allows,e.g., for the generation of increased crop yields and/or seed size inany of a wide variety of plants. An increase in crop yield can include,for example, increased leaf tissue, increased fruit, increased flowerproduction, increased root mass, and the like.

In particular embodiments of the invention, the variant CKI polypeptideis a KRP family member. This aspect of the present invention is based,at least in part, on the discovery that the CDK binding region of KRPfamily members is primarily responsible for inhibition of cyclin-CDKcomplexes. Thus, targeting the CDK binding region in KRP family members(as opposed to the cyclin binding region) allows for the generation ofmutant proteins that are particularly efficacious dominant negativeantagonists of wild-type CKI function.

Mutant CKI Polypeptides, Nucleic Acids, and Vectors

The CKI polypeptides of the present invention are mutant or variantproteins distinguishable from naturally occurring or wild-type CKIs. Thevariant CKI polypeptides comprise at least one modification, relative toa reference CKI polypeptide (e.g., a wild-type CKI protein), in the CDKor cyclin binding region of the protein. Typical modifications includeamino acid substitutions, insertions, and/or deletions as compared tothe corresponding wild-type sequence. Particularly suitablemodifications are amino acid substitutions. In certain embodiments, thevariant CKI polypeptide comprises at least one non-conservativemodification (e.g., a substitution).

The mutant CKI polypeptides of the present invention are dominantnegative proteins. The dominant negative approach is particularlyamenable to CKI polypeptides having two polypeptide regions separatelyinvolved in CDK and cyclin binding. The general approach for creatingthe dominant negative mutants of the present invention include modifyingone of the individual CDK and cyclin binding regions so as to modify“wild-type” CDK or cyclin binding. This modified CDK or cyclin bindingregion can result in either equivalent, reduced or eliminated binding tothe cyclin or CDK compared to the wild-type polypeptide. In either case,the mutation would reduce or eliminate the CKIs kinase inhibitoryactivity. In order to design a dominant negative CKI polypeptide thatcan interfere with the wild type function, the mutant polypeptide must(1) substantially bind to cyclin/CDK complexes; (2) not substantiallyinhibit the cyclin CDK complex even at high concentrations; and (3)compete with a wild-type CKI polypeptide for binding to the cyclin/CDKcomplex. In vivo, mutant CKI polypeptides that fulfill all of theserequirements result in elevated cyclin/CDK kinase activity in the cell,which in turn leads to increased cell proliferation and a higher mitoticindex that ultimately leads to plants with increased yield, largerseeds, and/or some other characteristic associated with an elevatedcyclin/CDK kinase activity in one or more regions of the plant.

In the particular case of KRP polypeptides, the CDK binding region,which is primarily responsible for cyclin/CDK kinase inhibition in thisfamily of CKI polypeptides, is targeted for modification.

Particularly suitable modifications include amino acid substitutions,insertions, or deletions. For example, amino acid substitutions can begenerated as modifications in the CDK or the cyclin-binding region thatreduce or eliminate binding. Similarly, amino acid substitutions can begenerated as modifications in the CDK or the cyclin-binding region thatreduces or eliminates the CKI inhibitory activity. In typicalembodiments, at least one non-conservative amino acid substitution,insertion, or deletion in the CDK or the cyclin binding region is madeto disrupt or modify binding of the CKI polypeptide to a CDK or cyclinprotein.

Substitutional CKI polypeptide mutants are those that have at least oneamino acid residue in a reference CKI protein sequence removed and adifferent amino acid inserted in its place at the same position. Thesubstitutions may be single, where only one amino acid in the moleculehas been substituted, or they may be multiple, where two or more aminoacids have been substituted in the same molecule. Substantial changes inthe activity of the CKI protein molecules of the present invention canbe obtained by substituting an amino acid with another whose side chainis significantly different in charge and/or structure from that of thenative amino acid, an amino acid with an opposite charge from that ofthe native amino acid, or an amino acid with the opposite hydrophilicityfrom that of the native amino acid, and the like. These types ofsubstitutions would be expected to affect the structure of thepolypeptide backbone and/or the charge or hydrophobicity of the moleculein the area of the substitution. In certain, exemplary embodiments ofthe present invention, a substitutional CKI mutant includes substitutionof a non-alanine residue with alanine. In other variations, thesubstitutional CKI includes substitution of an amino acid residue with,for example, an oppositely charged amino acid, an amino acid residuewith a larger side chain, an amino acid with the oppositehydrophilicity, a small non-polar amino acid (e.g., Cys, Thr, Ser, Ala,or Gly), or a polar amino acid (e.g., Pro, Glu, Asp, Asn, or Gln).

Insertional CKI polypeptide mutants are those with one or more aminoacids inserted immediately adjacent to an amino acid at a particularposition in the reference CKI protein molecule. Immediately adjacent toan amino acid means connected to either the α-carboxy or α-aminofunctional group of the amino acid. The insertion can be one or moreamino acids. The insertion can consist, e.g., of one or two conservativeamino acids. Amino acids similar in charge and/or structure to the aminoacids adjacent to the site of insertion are defined as conservative.Alternatively, mutant CKI include the insertion of an amino acid with acharge and/or structure that is substantially different from the aminoacids adjacent to the site of insertion.

Deletional CKI polypeptide mutants are those where one or more aminoacids in the reference CKI protein molecules have been removed. In someembodiments, deletional mutants will have one, two or more amino acidsdeleted in a particular region of the CKI protein molecule. Deletionalmutants can include, e.g., mutants having a truncation of the amino- orcarboxy-terminus.

The methods of the present invention can be applied to any recognizedmember of the CKI family of proteins in which individual regions areinvolved in binding of CDK and cyclin proteins to inhibit CDK function.In one embodiment, the mutant CKI proteins are mutations or variants ofproteins belonging to the KRP family of CKIs. As noted above, the CDKbinding region of KRP family members is primarily responsible for kinaseinhibition. Accordingly, the CDK binding region is preferably targetedfor modification in the design of variant KRP CKI polypeptides. The CDKbinding region of KRP proteins generally corresponds to amino acids145-168 of Brassica napus Krp1 (BnKrp1) (SEQ ID NO:17). In certainembodiments, modification of the CDK binding region of a KRP familymember comprises modification of at least one amino acid position, twoamino acid positions, or more within the region corresponding topositions 145-168 of BnKrp1. (The corresponding region in Arabidopsisthaliana Krp1 comprises amino acids 167-190.) Particularly suitableamino acid positions for modification include those positionscorresponding to amino acids 145, 148, 149, 151, 153, 155, 163, 164,165, and/or 167 of Brassica napus Krp1 (BnKrp1). The corresponding aminoacid residues in other CKI polypeptides are easily determined bysequence alignment using known methods and as further described herein.

In each case, the amino acids mentioned above are conserved in mammalianp27 and all contact the CDK in the crystal structure of p27 complexedwith Cyclin A and CDK2. In one embodiment, modification of the KRP CDKbinding region comprises modification of amino acids corresponding toamino acid positions 151 and 153 of BnKrp1 (e.g., an amino acidsubstitution, such as, for example, to alanine or an oppositely chargedamino acid, at each of these sites). In other exemplary variations, inaddition to modification of amino acids corresponding to positions 151and 153 of BnKrp1, modification of the KRP CDK binding region furtherincludes modification(s) at position(s) corresponding to amino acid(s)149, 164, and/or 165 of BnKrp1 (e.g., an additional amino acidsubstitution at a position corresponding to amino acid 149 of BnKrp1; ortwo additional amino acid substitutions at positions corresponding toboth amino acid 164 and amino acid 165 of BnKrp1). Such modification(s)to the KRP CKI polypeptide modify binding affinity for a CDK protein,while substantially preserving the ability of the variant protein tobind a cyclin protein.

Modification of the Krp CDK binding region of particular interestinclude, but are not limited to, modifications corresponding to any ofthe following amino acid substitutions:

BnKrp1 F145A; Y149A

BnKrp1 F145A; Y149A; F151A

BnKrp1 F145A; Y149A; F151A; F153A

BnKrp1 Y149A; F151A

BnKrp1 Y149A; F153A

BnKrp1 F151A; F153A

BnKrp1 F151A; F153A; Y149A

BnKrp1 F151A; F153A; E164A

BnKrp1 F151A; F153A; W165A

BnKrp1 F151A; F153A; E164A; W165A

BnKrp1 F151A; F153A; Y149A; E164A

BnKrp1 F151A; F153A; Y149A; W165A

BnKrp1 F151A; F153A; Y149A; E164A; W165A

BnKrp1 E164A; W165A

In certain embodiments, the CKI polypeptide modified as above is BnKrp1.In other variations, the CKI modified as above is not BnKrp1 (e.g.,Arabidopsis thaliana), with the substituted amino acids corresponding tothose set forth above.

Other modifications (e.g., substitutions, insertions, deletions) thataffect CDK or cyclin binding, including additional modifications in KRPfamily members, can be identified using a variety of techniques,including structural alignment methods, sequence alignment methods, andthe like. The mutant or variant proteins can be generated, for example,by using a PDA™ system previously described in U.S. Pat. Nos. 6,188,965;6,296,312; and 6,403,312; alanine scanning (see U.S. Pat. No.5,506,107), gene shuffling ((WO 01/25277), site saturation mutagenesis,mean field, sequence homology, or other methods known to those skill inthe art that guide the selection of point mutation sites and types, asdescribed further hereinbelow.

The CKI polypeptide variants of the present invention can be constructedby mutating the DNA sequences that encode the corresponding wild-typeCKI, or other corresponding CKI from which the variant is derived, suchas by using techniques commonly referred to as site-directedmutagenesis. Nucleic acid molecules encoding the CKI proteins can bemutated by a variety of polymerase chain reaction (PCR) techniques wellknown to one of ordinary skill in the art. (See, e.g., PCR Strategies(M. A. Innis, D. H. Gelfand, and J. J. Sninsky eds., 1995, AcademicPress, San Diego, Calif.) at Chapter 14); PCR Protocols: A Guide toMethods and Applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky, andT. J. White eds., Academic Press, NY, 1990). In addition, well knownchemical and/or radiation mutagenesis methods are well known in the artcan be used to induce mutations in the coding regions of KRP familymember proteins. Screening can be carried out to locate those plantsthat might comprise a desired nucleotide sequence encoding a mutant orvariant Krp of the present invention. Plants can be screened for changesin the amino acid sequence of the CKI, changes in kinase activity or theexpected changes in phenotype followed by sequence analysis.

By way of non-limiting example, the two primer system utilized in theTransformer Site-Directed Mutagenesis kit from Clontech, may be employedfor introducing site-directed mutants into a gene encoding a CKIprotein. Following denaturation of the target plasmid in this system,two primers are simultaneously annealed to the plasmid; one of theseprimers contains the desired site-directed mutation, the other containsa mutation at another point in the plasmid resulting in elimination of arestriction site. Second strand synthesis is then carried out, tightlylinking these two mutations, and the resulting plasmids are transformedinto a mutS strain of E. coli. Plasmid DNA is isolated from thetransformed bacteria, restricted with the relevant restriction enzyme(thereby linearizing the unmutated plasmids), and then retransformedinto E. coli. This system allows for generation of mutations directly inan expression plasmid, without the necessity of subcloning or generationof single-stranded phagemids. The tight linkage of the two mutations andthe subsequent linearization of unmutated plasmids results in highmutation efficiency and allows minimal screening. Following synthesis ofthe initial restriction site primer, this method requires the use ofonly one new primer type per mutation site. Rather than prepare eachpositional mutant separately, a set of “designed degenerate”oligonucleotide primers can be synthesized in order to introduce all ofthe desired mutations at a given site simultaneously. Transformants canbe screened by sequencing the plasmid DNA through the mutagenized regionto identify and sort mutant clones. Each mutant DNA can then berestricted and analyzed by electrophoresis, such as for example, on aMutation Detection Enhancement gel (J. T. Baker) to confirm that noother alterations in the sequence have occurred (by band shiftcomparison to the unmutagenized control). Alternatively, the entire DNAregion can be sequenced to confirm that no additional mutational eventshave occurred outside of the targeted region.

The verified mutant duplexes in the pET (or other) overexpression vectorcan be employed to transform E. coli such as strain E. coli BL21 (DE3)pLysS, for high level production of the mutant protein, and purificationby standard protocols. The method of FAB-MS mapping can be employed torapidly check the fidelity of mutant expression. This technique providesfor sequencing segments throughout the whole protein and provides thenecessary confidence in the sequence assignment. In a mapping experimentof this type, protein is digested with a protease (the choice willdepend on the specific region to be modified since this segment is ofprime interest and the remaining map should be identical to the map ofunmutagenized protein). The set of cleavage fragments is fractionatedby, for example, microbore HPLC (reversed phase or ion exchange, againdepending on the specific region to be modified) to provide severalpeptides in each fraction, and the molecular weights of the peptides aredetermined by standard methods, such as FAB-MS. The determined mass ofeach fragment are then compared to the molecular weights of peptidesexpected from the digestion of the predicted sequence, and thecorrectness of the sequence quickly ascertained. Since this mutagenesisapproach to protein modification is directed, sequencing of the alteredpeptide should not be necessary if the MS data agrees with prediction.If necessary to verify a changed residue, CAD-tandem MS/MS can beemployed to sequence the peptides of the mixture in question, or thetarget peptide can be purified for subtractive Edman degradation orcarboxypeptidase Y digestion depending on the location of themodification.

In the design of a particular site directed mutagenesis, it is generallydesirable to first make a non-conservative substitution and determine(a) whether the targeted CDK- or cyclin-binding activity is impaired and(b) whether any non-targeted activity (e.g., cyclin binding if theCDK-binding region is targeted) is greatly impaired as a consequence. Ifthe residue is by this means demonstrated to be important to anon-targeted biological activity, then conservative substitutions can bemade.

Other site directed mutagenesis techniques can also be employed with CKInucleotide sequences. For example, restriction endonuclease digestion ofDNA followed by ligation may be used to generate deletion variants ofCKIs, as described generally in section 15.3 of Sambrook et al.,Molecular Cloning: A Laboratory Manual (2nd Ed., 1989 Cold Spring HarborLaboratory Press, New York, N.Y.). A similar strategy may be used toconstruct insertion variants, as described in section 15.3 of Sambrooket al., supra. More recently Zhu et al. (Proc. Natl. Acad. Sci. USA96:8768-8773, 1999) have devised a method of targeting mutations toplant genes in vivo using chimeric RNA/DNA oligonucleotides.

Mutant polypeptides with more than one amino acid substituted can begenerated in one of several ways. If the amino acids are located closetogether in the polypeptide chain, they may be mutated simultaneouslyusing one oligonucleotide that codes for all of the desired amino acidsubstitutions. If however, the amino acids are located some distancefrom each other (separated by more than ten amino acids, for example) itis more difficult to generate a single oligonucleotide that encodes allof the desired changes. Instead, one of two alternative methods may beemployed. In the first method, a separate oligonucleotide is generatedfor each amino acid to be substituted. The oligonucleotides are thenannealed to the single-stranded template DNA simultaneously, and thesecond strand of DNA that is synthesized from the template will encodeall of the desired amino acid substitutions. An alternative methodinvolves two or more rounds of mutagenesis to produce the desiredmutant. The first round is as described for the single mutants:wild-type CKI DNA is used for the template, an oligonucleotide encodingthe first desired amino acid substitution(s) is annealed to thistemplate, and the heteroduplex DNA molecule is then generated. Thesecond round of mutagenesis utilizes the mutated DNA produced in thefirst round of mutagenesis as the template. Thus, this template alreadycontains one or more mutations. The oligonucleotide encoding theadditional desired amino acid substitution(s) is then annealed to thistemplate, and the resulting strand of DNA now encodes mutations fromboth the first and second rounds of mutagenesis. This resultant DNA canbe used as a template in a third round of mutagenesis, and so on.

One particularly suitable technique for guiding identification ofsuitable modifications includes aligning CKI proteins by sequencealignment. There are a number of sequence alignment methodologiesdiscussed above that may be used. Sequence-based alignment programsinclude, for example, Smith-Waterman searches, Needleman-Wunsch, DoubleAffine Smith-Waterman, frame search, Gribskov/GCG profile search,Gribskov/GCG profile scan, profile frame search, Bucher generalizedprofiles, Hidden Markov models, Hframe, Double Frame, Blast, Psi-Blast,Clustal, and GeneWise. (See, e.g., Altschul et al., J. Mol. Biol.215:403-410, 1990; Altschul et al., Nucleic Acids Res. 25:3389-3402,1997, both incorporated by reference).

The amino acid sequences of related CKI polypeptides can be aligned, forexample, into a multiple sequence alignment (MSA). (See, e.g., FIG. 1.)The MSA can also be used to extend structural information known for oneor more CKI polypeptides to additional CKI polypeptides (typically toCKI polypeptides sharing substantial sequence identity) that may no yethave been identified. Due to the high extent of structural homologybetween different CKI polypeptides, the MSA can be used as a reliablepredictor of the effects of modifications at various positions withinthe alignment. Accordingly, in the case of KRP family members, forexample, the CKI sequence and numbering shown in FIG. 1 can be used asan MSA reference point for any other KRP family member protein sequence.As noted previously, particularly suitable amino acid positions formodification include those corresponding to amino acid positions 145,148, 149, 151, 153, 155, 163, 164, 165 and/or 167 of Brassica napus KRP1(BnKrp1). Using the alignment depicted in FIG. 1, and/or using alignmentprograms known in the art such as those described herein, one can use asa reference point the numbering system of the alignment program and maycorrelate the relevant positions of the CKI polypeptide with equivalentpositions in other recognized members of CKIs or structural homologuesand families. Similar methods can be used for alignment of the aminoacid sequence(s) of CKI polypeptide that have yet to be sequenced.

In certain cases, the amino acids in the CKI polypeptide that interactwith a CDK or cyclin protein can be identified directly from athree-dimensional structure of a CKI/CDK or CKI/cyclin complex.Equivalent information can be derived by analysis of the CKI/CDK orCKI/cyclin complex of a related CKI polypeptide. Thus, structuralalignments can be used to generate the variant CKI polypeptides of theinvention. There are a wide variety of structural alignment programsknown in the art. See, e.g., VAST from the NCBI website; SSAP (Orengoand Taylor, Methods Enzymol. 266:617-635, 1996); SARF2 (Alexandrov,Protein Eng. 9:727-732, 1996) CE (Shinydyalov and Bourne, Protein Eng.11:739-747, 1998); (Orengo et al., Structure 5:1093-108, 1997; Dali(Holm et al., Nucl. Acid Res. 26:316-9, 1998, all of which areincorporated by reference).

Accordingly, useful modifications at CKI/CDK or CKI/cyclin interfacesmay be selected using protein design or modeling algorithms such as PDA™technology (see U.S. Pat. Nos. 6,188,965; 6,269,312; and 6,403,312,hereby incorporated by reference). Algorithms in this class generallyuse atomic-level or amino acid level scoring functions to evaluate thecompatibility of amino acid sequences with the overall tertiary andquaternary structure of a protein. Thus, algorithms of this class can beused to select CDK- or cyclin-binding modifications and/or disruptionsthat do not substantially perturb the ability of variant CKI proteins toproperly fold and interact with naturally occurring targetscorresponding to non-modified regions of the protein. These technologiestypically use high-resolution structural information of the targetprotein as input. In one embodiment, an experimentally determinedstructure of the appropriate CKI protein is used as input. Inalternative embodiments, a MSA can be used to guide the construction ofatomic-level homology models for CKI members based on the subset of thefamily whose three-dimensional structures have been determined usingcrystallographic or related methods. In yet another embodiment, thestructural model of mammalian p27/cyclin interface can be used topredict the contact amino acid residue for plant CKI.

Mutant CKI polypeptides having modified, for example, reduced bindingfor a CDK or cyclin protein can also be identified by a large variety ofother methods, including, for example, directed evolution (e.g., errorprone PCR, DNA shuffling, and the like), single-site saturationmutagenesis, and alanine-scanning mutagenesis. In the case of KRP CKIs,for example, the use of these and/or other methods can allow theidentification of additional modifications that reduce CDK bindingactivity and that lie outside of the CDK binding region describedherein.

Also, molecular dynamics calculations can be used to computationallyscreen sequences by individually calculating mutant sequence scores andcompiling a list. Also, residue pair potentials can be used to scoresequences (Miyazawa et al., Macromolecules 18: 534-552, 1985,incorporated by reference herein) during computational screening.

Alternatively, libraries of variant CKI proteins can be made fortesting. For example, a library of variant CKI amino acid sequences maybe used to design nucleic acids encoding the variant CKI sequences andwhich may then be cloned into host cells, expressed, and assayed. Thechoice of codons, suitable expression vectors, and suitable host cellswill typically vary depending on a number of factors, and can be easilyoptimized as needed.

In one particularly suitable method for screening libraries of mutantstesting for dominant negative antagonist activity and/or other desiredactivities as outlined herein, multiple PCR reactions with pooledoligonucleotides are performed. Overlapping oligonucleotides aresynthesized that correspond to the full-length gene. Theseoligonucleotides may represent all of the different amino acids at eachvariant position or subsets. These oligonucleotides may be pooled inequal proportions and multiple PCR reactions performed to createfull-length sequences containing the combinations of mutations definedby the library. In addition, this may be done using error-prone PCRmethods.

Typically, each overlapping oligonucleotide comprises only one positionto be varied. Alternatively, the variant positions are too closetogether to allow this and multiple variants per oligonucleotide areused to allow complete recombination of all the possibilities (i.e.,each oligo may contain the codon for a single position being mutated, orfor more than one position being mutated). The multiple positions beingmutated are preferably close in sequence to prevent the oligo nucleotidelength from being impractical. For mutating multiple positions on anoligonucleotide, particular combinations of mutations may be included orexcluded in the library by including or excluding the oligonucleotideencoding that combination. For example, as discussed herein, there maybe correlations between variable regions; that is, when position X is acertain residue, position Y must (or must not) be a particular residue.These sets of variable positions are sometimes referred to herein as a“cluster.” When the clusters are comprised of residues close together,and thus can reside on one oligonucleotide primer, the clusters can beset to the “good” correlations, and eliminate the bad combinations thatmay decrease the effectiveness of the library. However, if the residuesof the cluster are far apart in sequence, and thus will reside ondifferent oligonucleotides for synthesis, it may be desirable to eitherset the residues to the “good” correlation, or eliminate them asvariable residues entirely. Alternatively, the library may be generatedin several steps, so that the cluster mutations only appear together.This procedure, i.e., the procedure of identifying mutation clusters andeither placing them on the same oligonucleotides or eliminating themfrom the library or library generation in several steps preservingclusters, can considerably enrich the library with properly foldedprotein. Identification of clusters may be carried out by a number ofways, e.g., by using known pattern recognition methods, comparisons offrequencies of occurrence of mutations or by using energy analysis ofthe sequences to be experimentally generated (for example, if the energyof interaction is high, the positions are correlated). Thesecorrelations may be positional correlations (e.g., variable positions 1and 2 always change together or never change together) or sequencecorrelations (e.g., if there is residue A at position 1, there is alwaysresidue B at position 2). (See Pattern Discovery in Biomolecular Data:Tools, Techniques, and Applications; (Jason T. L. Wang, Bruce A.Shapiro, Dennis Shasha eds., New York, Oxford University, 1999);Andrews, Introduction to Mathematical Techniques in Pattern Recognition(New York, Wiley-Interscience, 1972); Applications of PatternRecognition (K. S. Fu ed., Boca Raton, Fla., CRC Press, 1982); GeneticAlgorithms for Pattern Recognition (Sankar K. Pal and Paul P. Wang eds.,Boca Raton, Fla., CRC Press, 1996); Pandya, Pattern Recognition withNeural Networks in C++ (Boca Raton, Fla., CRC Press, 1996; Handbook ofPattern Recognition & Computer Vision (C. H. Chen, L. F. Pau, and P. S.P. Wang. eds., 2nd ed. Singapore; River Edge, N.J., World Scientific,c1999); Friedman, Introduction to Pattern Recognition: Statistical,Structural, Neural, and Fuzzy Logic Approaches (River Edge, N.J., WorldScientific, 1999, Series title: Series in machine perception andartificial intelligence; vol. 32); all of which are expresslyincorporated by reference. In addition, programs used to search forconsensus motifs can be used as well.

Oligonucleotides with insertions or deletions of codons can be used tocreate a library expressing different length proteins. In particular,computational sequence screening for insertions or deletions may resultin secondary libraries defining different length proteins, which can beexpressed by a library of pooled oligonucleotide of different lengths.

In another embodiment, mutant CKI polypeptides of the invention arecreated by shuffling a family (e.g., a set of mutants); that is, someset of the top sequences (if a rank-ordered list is used) can beshuffled, either with or without error-prone PCR. “Shuffling” in thiscontext means a recombination of related sequences, generally in arandom way. It can include “shuffling” as defined and exemplified inU.S. Pat. Nos. 5,830,721; 5,811,238; 5,605,793; 5,837,458 and PCTUS/19256, all of which are incorporated by reference herein. This set ofsequences may also be an artificial set; for example, from a probabilitytable (for example generated using SCMF) or a Monte Carlo set.Similarly, the “family” can be the top 10 and the bottom 10 sequences,the top 100 sequence, and the like. This may also be done usingerror-prone PCR.

Thus, in silico shuffling can be performed using the computationalmethods described herein (e.g., starting with two libraries or twosequences, random recombinations of the sequences may be generated andevaluated).

Error-prone PCR can be performed to generate a library of variant CKIpolypeptides. See U.S. Pat. Nos. 5,605,793, 5,811,238, and 5,830,721,which are hereby incorporated by reference. This can be done on theoptimal sequence or on top members of the library, or some otherartificial set or family. In this method, the gene for the optimalsequence found in the computational screen of the primary library can besynthesized. Error-prone PCR is then performed on the optimal sequencegene in the presence of oligonucleotides that code for the mutations atthe variant positions of the library (bias oligonucleotides). Theaddition of the oligonucleotides will create a bias favoring theincorporation of the mutations in the library. Alternatively, onlyoligonucleotides for certain mutations can be used to bias the library.

Gene shuffling can be performed with error-prone PCR on the gene for theoptimal sequence, in the presence of bias oligonucleotides, to create aDNA sequence library that reflects the proportion of the mutations foundin the CKI library. The choice of the bias oligonucleotides can be donein a variety of ways; they can chosen on the basis of their frequency,e.g., oligonucleotides encoding high mutational frequency positions canbe used; alternatively, oligonucleotides containing the most variablepositions can be used, such that the diversity is increased; if thesecondary library is ranked, some number of top scoring positions can beused to generate bias oligonucleotides; random positions may be chosen;a few top scoring and a few low scoring ones may be chosen; and thelike. What is important is to generate new sequences based on preferredvariable positions and sequences.

In another variation, PCR using a wild-type gene or other gene can beused. In this embodiment, a starting gene is used (e.g., the wild-typegene, the gene encoding the global optimized sequence, or a consensussequence obtained, e.g., from aligning homologous sequences fromdifferent organisms). In this embodiment, oligonucleotides are used thatcorrespond to the variant positions and contain the different aminoacids of the library. PCR is done using PCR primers at the termini. Thisprovides two benefits. First, this generally requires feweroligonucleotides and may result in fewer errors. Second, it hasexperimental advantages in that if the wild-type gene is used, it neednot be synthesized.

The mutant CKI polypeptides can be from any number of organisms, withCKI polypeptides from plants being particularly preferred. Suitableplants include, e.g., the class of higher plants amenable totransformation techniques, including both monocotyledonous anddicotyledonous plants, as well as certain lower plants such as algae. Itincludes plants of a variety of ploidy levels, including polyploid,diploid and haploid. In specific embodiments, the plant is Brassicanapus, Arabidopsis thaliana, Glycine max, maize, rice, wheat, alfalfa,cotton, poplar, and the like.

As described above, the mutant CKI polypeptides of the invention aredominant negative antagonists of wild-type CKI polypeptides. In certainembodiments, the mutant CKI polypeptide physically interacts with onlyone or both of its corresponding cyclin or CDK proteins (i.e., theendogeneous, naturally occurring CDK or cyclin protein from the speciesfrom which the CKI mutant was derived) such that a CDK/cyclin complexcomprising the mutant CKI is protected for CDK/cyclin kinase inhibitionby the wild-type CKI protein.

In an alternative, non-mutually exclusive embodiment, the mutant CKIpolypeptide physically interacts with only one or both of a heterologousCDK or cyclin proteins (i.e., a naturally occurring CDK or cyclinprotein from a species that is different from the species from which theCKI mutant was derived), such that a complex of the heterologousCDK/cyclin comprising the mutant CKI is protected from CDK/cyclin kinaseinhibition by a wild-type CKI protein that corresponds to the CDK andcyclin proteins within the complex (i.e., by a wild-type CKI proteinendogenous to the CDK and cyclin proteins).

In some embodiments, the mutant CKI polypeptides of the invention arehighly specific antagonists for the corresponding wild-type CKI protein.In alternative embodiments, the mutant CKI polypeptides of the inventionare specific antagonists for more than one wild-type CKI polypeptide.For example, a mutant Arabidopsis CKI polypeptide may be a specificantagonist of a wild-type Arabidopsis CKI polypeptide only, or specificfor wild-type Arabidopsis, Brassica napus, Glycine max, maize, rice,cotton, and/or poplar CKI polypeptides. Also, a mutant Brassica CKIpolypeptide may be a specific antagonist of a wild-type Brassica CKIpolypeptide only, or is a specific antagonist for wild-type Arabidopsis,Brassica napus, Glycine max, maize, rice, wheat, alfalfa, cotton, and/orpoplar.

Mutant CKI polypeptides exhibit substantially decreased biologicalactivity as compared to wild-type CKI polypeptides, including, e.g.,modified binding to one of a CDK or cyclin protein and decreasedinhibition of CDK/cyclin kinase complexes. Such decreased biologicalactivity can be tested and validated using in vivo and/or in vitroassays. Suitable assays include, but are not limited to, CDK/cyclinkinase activity assays; CDK or cyclin binding assays; and cellularproliferation assays. A substantial decrease in biological activity ascompared to wild-type CKI polypeptides means that the biologicalactivity of the variant CKI polypeptide is less than 80% or less than70%, typically less than 60% or less than 50%, more typically less than40% or less than 30%, and preferably less than 20% or less than 10% thatof a corresponding wild-type CKI polypeptide.

In some embodiments, the mutant CKI polypeptide includes a modification,as compared to a wild-type CKI polypeptide, in addition to thoseoutlined herein (i.e., the mutant CKI proteins may contain additionalmodifications as compared to a corresponding wild-type CKI protein otherthan those used to generate dominant negative proteins). Examplesinclude, but are not limited to, amino acid substitutions introduced toenable soluble expression in E. coli and amino acid substitutionsintroduced to optimize solution behavior. In addition, as outlinedherein, any of the mutations depicted herein can be combined in any wayto form additional variant CKI polypeptides. In addition, mutant CKIpolypeptides can be made that are longer than the correspondingwild-type protein, for example, by the addition of epitope orpurification tags, the addition of other fusion sequences, and the likeor they may be shorter.

Mutant CKI polypeptides can also be identified as being encoded bymutant CKI nucleic acids. In the case of the nucleic acid, the overallsequence identity of the nucleic acid sequence is commensurate withamino acid sequence identity but takes into account the degeneracy inthe genetic code and codon bias of different organisms. Accordingly, thenucleic acid sequence identity may be either lower or higher than thatof the protein sequence, with lower sequence identity being typical.

As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids may be made,all of which encode the mutant CKI polypeptides of the presentinvention. Thus, having identified a particular amino acid sequence, anynumber of different nucleic acids encoding the mutant protein can bemade by simply modifying the sequence of one or more codons in a waywhich does not change the amino acid sequence of the mutant CKIpolypeptide.

The mutant CKI polypeptides and nucleic acids of the present inventionare preferably recombinant (unless made synthetically). As noted supra,mutants are typically prepared by site-specific mutagenesis ofnucleotides in the DNA encoding a corresponding CKI protein, usingcassette or PCR mutagenesis or another technique well known in the art,to produce DNA encoding the mutant, and thereafter expressing the DNA inrecombinant cell culture. Amino acid sequence mutants are characterizedby the predetermined nature of the mutation, a feature that sets themapart from naturally occurring allelic or interspecies variation of themutant CKI protein amino acid sequence. As outlined above, the variantstypically exhibit similar binding of either a CDK or cyclin protein(accordingly, as compared to a corresponding wild-type CKI protein, themutant exhibits a substantial decrease in its inhibitory activity;although variants can also be selected that have additional variantcharacteristics.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed variant CKI proteins screenedfor the optimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well-known, for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants is done using assays of mutant CKIprotein activities for optimum characteristics.

In some embodiments, amino acid substitutions are of single residues. Inother embodiments, multiple amino acid residues are substituted (e.g.,2, 3, 4, or more amino acids can be substituted). Insertions aretypically on the order of from about 1 to 20 about amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, or from about 1 to about 30 residues,although in some cases deletions may be much larger. In certainembodiments, mutant CKI polypeptides are chimeras derived from two ormore wild-type CKI proteins, with at least one modification in the CDKor cyclin binding region as outlined herein.

Substitutions, deletions, insertions, or any combination thereof, areused to arrive at a final mutant. Generally, the modification(s) aredone with respect to relatively few amino acids to minimize thealteration of the molecule. However, larger changes may be tolerated incertain circumstances.

Using a nucleic acid of the present invention encoding a mutant CKIpolypeptide, a variety of vectors can be made. Any vector containingreplicon and control sequences that are derived from a speciescompatible with the host cell can be used in the practice of theinvention. The vectors can be either self-replicating extrachromosomalvectors or vectors which integrate into a host genome. Generally,expression vectors include transcriptional and translational regulatorynucleic acid regions operably linked to the nucleic acid encoding themutant CKI polypeptide. The term “control sequences” refers to DNAsequences necessary for the expression of an operably linked codingsequence in a particular host organism. The control sequences that aresuitable for prokaryotes, for example, include a promoter, optionally anoperator sequence, and a ribosome binding site. The transcriptional andtranslational regulatory nucleic acid regions will generally beappropriate to the host cell used to express the polypeptide.

In certain embodiments, CKI nucleic acids are modified for enhance geneexpression in a host cell using codon optimization, which involvesreplacing a codon sequence with a translation codon (corresponding tothe same amino acid) that is highly used in the species of cell in whichthe DNA molecule is to be expressed. Examples of BnKrp1 mutant codingsequences (BnKrp1 F151A; F153A and Y149A; F151A; F153A) that have beenoptimized for expression in Maize are shown in FIG. 3. Codonoptimization procedures are generally well-known in the art and can beused by those skilled in the art to obtain other codon optimized mutantKrp1 sequences in accordance with the present invention.

Numerous types of appropriate expression vectors, and suitableregulatory sequences are known in the art for a variety of host cells.In general, the transcriptional and translational regulatory sequencesmay include, e.g., promoter sequences, ribosomal binding sites,transcriptional start and stop sequences, translational start and stopsequences, and enhancer or activator sequences. In typical embodiments,the regulatory sequences include a promoter and transcriptional startand stop sequences. Vectors also typically include a polylinker regioncontaining several restriction sites for insertion of foreign DNA. Theconstruction of suitable vectors containing DNA encoding replicationsequences, regulatory sequences, phenotypic selection genes, and thevariant CKI DNA of interest are prepared using standard recombinant DNAprocedures. Isolated plasmids, viral vectors and DNA fragments arecleaved, tailored, and ligated together in a specific order to generatethe desired vectors, as is well-known in the art (see, e.g., Maniatis,supra, and Sambrook et al., supra).

Promoter sequences encode either constitutive or inducible promoters.The promoters can be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention. In a typical embodiment, the promoters are strong promoters,allowing high expression in cells, particularly plant cells. Promotersparticularly suitable for use in accordance with the present inventionare described further infra.

In addition, the expression vector can comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in plant orinsect cells for expression and in a prokaryotic host for cloning andamplification. Further, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences which flank theexpression construct. The integrating vector can be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In certain embodiments, the expression vector contains a selectablemarker gene to allow the selection of transformed host cells. Selectiongenes are well known in the art and will vary with the host cell used.Suitable selection genes can include, for example, genes coding forampicillin and/or tetracycline resistance, which enables cellstransformed with these vectors to grow in the presence of theseantibiotics.

In one aspect of the present invention, a mutant CKI nucleic acid isintroduced into a cell, either alone or in combination with a vector. By“introduced into” or grammatical equivalents herein is meant that thenucleic acids enter the cells in a manner suitable for subsequentintegration, amplification, and/or expression of the nucleic acid. Themethod of introduction is largely dictated by the targeted cell type.Exemplary methods include CaPO₄ precipitation, liposome fusion,lipofectin®, electroporation, viral infection, and the like. Methodsparticularly suitable for introduction into plant cells are known in theart and are also described infra (see Section II, “Transgenic Plants”).Such methods include, for example, bombardment of cells with DNA ladenmicroprojectiles. The mutant CKI nucleic acids may stably integrate intothe genome of the host cell, or may exist either transiently or stablyin the cytoplasm (e.g., through the use of traditional plasmids,utilizing standard regulatory sequences, selection markers, and thelike).

Prokaryotes can be used as host cells for the initial cloning steps ofthe present invention. They are particularly useful for rapid productionof large amounts of DNA, for production of single-stranded DNA templatesused for site-directed mutagenesis, for screening many mutantssimultaneously, and for DNA sequencing of the mutants generated.Suitable prokaryotic host cells include E. coli K12 strain 94 (ATCC No.31,446), E. coli strain W3110 (ATCC No. 27,325) E. coli X1776 (ATCC No.31,537), and E. coli B; however many other strains of E. coli, such asHB11, JM101, NM522, NM538, NM539, and many other species and genera ofprokaryotes including bacilli such as Bacillus subtilis, otherenterobacteriaceae such as Salmonella typhimurium or Serratia marcesans,and various Pseudomonas species can all be used as hosts. Prokaryotichost cells or other host cells with rigid cell walls are typicallytransformed using the calcium chloride method as described in section1.82 of Sambrook et al., supra. Alternatively, electroporation can beused for transformation of these cells. Prokaryote transformationtechniques are set forth in for example Dower, in Genetic Engineering,Principles and Methods 12:275-296 (Plenum Publishing Corp., 1990);Hanahan et al., Meth. Enymol., 204:63, 1991. Plasmids typically used fortransformation of E. coli include pBR322, pUCI8, pUCI9, pUCI8, pUCI9,and Bluescript M13, all of which are described in sections 1.12-1.20 ofSambrook et al., supra. However, many other suitable vectors areavailable as well.

The mutant CKI polypeptides of the present invention are typicallyproduced by culturing a host cell transformed with an expression vectorcontaining a nucleic acid encoding the mutant CKI polypeptide, under theappropriate conditions to induce or cause expression of the mutant CKIpolypeptide. For modulation of cell division, the CKI protein isexpressed in its normal intracellular form. For applications of thepresent invention that include harvest or isolation of a mutant CKIpolypeptide, the CKI mutant can be expressed as an intracellular proteinor, alternatively, in a form that is secreted from the host cell. Manyeukaryotic proteins normally secreted from the cell contain anendogenous secretion signal sequence as part of the amino acid sequence.Thus, proteins normally found in the cytoplasm can be targeted forsecretion by linking a signal sequence to the protein. This is readilyaccomplished by ligating DNA encoding a signal sequence to the 5′ end ofthe DNA encoding the protein and then expressing this fusion protein inan appropriate host cell. The DNA encoding the signal sequence can beobtained as a restriction fragment from any gene encoding a protein witha signal sequence. Thus, prokaryotic, yeast, and eukaryotic signalsequences can be used herein, depending on the type of host cellutilized to practice the invention. The DNA and amino acid sequenceencoding the signal sequence portion of several eukaryotic genesincluding, for example, human growth hormone, proinsulin, and proalbuminare known (see Stryer, Biochemistry (W.H. Freeman and Company, New York,N.Y., 1988, p. 769)), and can be used as signal sequences in appropriateeukaryotic host cells. Yeast signal sequences, such as, for example,acid phosphatase (Arima et al., Nuc. Acids Res. 11:1657, 1983),α-factor, alkaline phosphatase and invertase may be used to directsecretion from yeast host cells. Prokaryotic signal sequences from genesencoding, for example, LamB or OmpF (Wong et al., Gene 68:193, 1988),MalE, PhoA, or beta-lactamase, as well as other genes, may be used totarget proteins expressed in prokaryotic cells into the culture medium.

The conditions appropriate for mutant CKI polypeptide expression willvary with the choice of the expression vector and the host cell, andwill be easily ascertained by one skilled in the art through routineexperimentation. For example, the use of constitutive promoters in theexpression vector will require optimizing the growth and proliferationof the host cell, while the use of an inducible promoter requires theappropriate growth conditions for induction. In addition, in someembodiments, the timing of the harvest is important. For example, thebaculoviral systems used in insect cell expression are lytic viruses,and thus harvest time selection can be important for product yield.

The promoters most commonly used in prokaryotic vectors include thep-lactamase (penicillinase) and lactose promoter systems (Chang et al.,Nature 375:615, 1978; Itakura et al., Science 198:1056, 1977; Goeddel etal., Nature 281:544, 1979) and a tryptophan (trp) promoter system(Goeddel et al., Nucl. Acids Res. 8:4057, 1980; EPO Appl. Publ. No.36,776), and the alkaline phosphatase systems. While these are the mostcommonly used, other microbial promoters have been utilized, and detailsconcerning their nucleotide sequences have been published, enabling askilled worker to ligate them functionally into plasmid vectors (seeSiebenlist et al., Cell 20:269, 1980).

An illustrative example of a responsive promoter system that can be usedin the practice of this invention is the glutathione-5-transferase (GST)system in maize. GSTs are a family of enzymes that can detoxify a numberof hydrophobic electrophilic compounds that often are used aspre-emergent herbicides (Weigand et al., Plant Molecular Biology7:235-243, 1986). Studies have shown that the GSTs are directly involvedin causing this enhanced herbicide tolerance. This action is primarilymediated through a specific 1.1 kb mRNA transcription product. In short,maize has a naturally occurring quiescent gene already present that canrespond to external stimuli and that can be induced to produce a geneproduct. This gene has previously been identified and cloned. Thus, inone embodiment of this invention, the promoter is removed from the GSTresponsive gene and attached to a mutant CKI coding sequence. If themutant CKI gene is derived from a genomic DNA source than it isnecessary to remove the native promoter during construction of thechimeric gene. This engineered gene is the combination of a promoterthat responds to an external chemical stimulus and a gene responsiblefor successful production of a mutant CKI protein.

An inducible promoter is a promoter that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer the DNAsequences or genes will not be transcribed. Typically the proteinfactor, that binds specifically to an inducible promoter to activatetranscription, is present in an inactive form which is then directly orindirectly converted to the active form by the inducer. The inducer canbe a chemical agent such as a protein, metabolite, a growth regulator,herbicide or a phenolic compound or a physiological stress imposeddirectly by heat, cold, salt, or toxic elements or indirectly throughthe action of a pathogen or disease agent such as a virus. A plant cellcontaining an inducible promoter can be exposed to an inducer byexternally applying the inducer to the cell or plant such as byspraying, watering, heating or similar methods. If it is desirable toactivate the expression of the target gene to a particular time duringplant development, the inducer can be so applied at that time.

Examples of such inducible promoters include heat shock promoters, suchas the inducible 70 KD heat shock promoter of Drosphilia melanogaster(Freeling et al., Ann. Rev. of Genetics 19:297-323); a cold induciblepromoter, such as the cold inducible promoter from B. napus (White etal., Plant Physiol. 106, 1994); and the alcohol dehydrogenase promoterwhich is induced by ethanol (Nagao et al., Surveys of plant Molecularand Cell Biology Vol. 3, p 384-438 (B. J. MifIin ed., Oxford UniversityPress, Oxford, 1986).

A constitutive promoter is a promoter that is capable of directly orindirectly activating the transcription of one or more DNA sequences orgenes in all tissues of a transgenic plant. Typically, a constitutivepromoter such as the 35 S promoter of CaMC (Odell, Nature 313:810-812,1985) is used. Other examples of constitutive promoters useful in plantsinclude the rice actin promoter (Elroy et al, Plant Cell 2:163-171,1990), maize HE histone (Lepetit et al., Mol Gen. Genet. 231:276-285,1992) and the like.

The CKI transgenes of the present invention can be expressed using apromoter such as is the BCEA (B. campestris embryo) promoter which hasbeen shown to direct high levels of expression in very early seeddevelopment (i.e., is transcribed before the napin promoter). This is aperiod prior to storage product accumulation but of rapid pigmentbiosynthesis in the Brassica seed (derived from Johnson-Flanagan et al.,J. Plant Physiol. 136:180, 1989; Johnson-Flanagan et al., Physiol. Plant81:301, 1991). Seed storage protein promoters have also been shown todirect a high level of expression in a seed-specific manner (Voelker etal., Plant Cell 1:95, 1989; Altenbach et al, Plant Mol. Biol. 13:513,1989; Lee et al., Proc. Natl. Acad. Sci. USA 99:6181, 1991; Russell etal., Transgenic Res. 6:157-68, 1997). The napin promoter has been shownto direct oleosin gene expression in transgenic Brassica, such thatoleosin accumulates to approximately 1% of the total seed protein (Leeet al., Proc. Natl. Acad. Sci. USA 99:6181, 1991). In choosing apromoter, it may be desirable to use a tissue-specific ordevelopmentally regulated promoter that allows suppression oroverexpression in certain tissues without affecting expresssion in othertissues. “Tissue specific promoters” refer to coding regions that directgene expression primarily in specific tissues such as, e.g., roots,leaves, stems, pistils, anthers, flower petals, seed coat, seednucellus, or epidermal layers. Transcription stimulators, enhancers oractivators can be integrated into tissue specific promoters to create apromoter with a high level of activity that retains tissue specificity.For instance, promoters utilized in overexpression will preferably betissue-specific. Overexpression in the wrong tissue such as leaves, whenattempting to overexpress in seed storage areas, could be deleterious.Particularly suitable promoters are those that allow for exampleseed-specific, root specific, leaf specific, fruit specific expression,and the like. This can be especially useful since seeds, roots, leavesand fruit are of particular interest. Some promoters specific fordifferent tissue types are already available or can be isolated bywell-established techniques (see, e.g., U.S. patent Nos. 5,792,925;5,783,393; 5,859,336; 5,866,793; 5,898,096; and 5,929,302) and asfurther described below. Table 1 lists other embryo specific promotersthat can be used to practice the present invention. TABLE 1 EmbryoSpecific Promoters Promoter Embryo Endosperm Timing Reference oleosinfrom strong, none traces at heart, Al et al., Plant Mol. Arabidopsisuniform higher early- to Biol. 25: 193-205, 1994. late-cotyledonarystage USP from strong, uniform none early not known, Baumlein et al.,Mol. Vicia faba strong in late cot. Gen. Genet. 225: 459-467, 1991.Legumin from strong, aleurone early not known, Baumlein et al., supraVicia faba preferential in layer (late) strong in late cot. 1991.cotyledons Napin from ? late Kohno-Murase, Plant Brassica Mol. Biol. 26:1115-1124, 1994 Albumin S1 in axis only none early- to late- Guerche etal., Plant from cotyledonary Cell 2: 469-478, 1990 Arabidopsis stageAlbumin S2 in axis and none early- to late- Guerche et al., supra,cotyledons cotyledonary 1990. stage

In particular, embodiments to the present invention, a seed specificpromoter that is particularity active during the development of theembryonic plant of an immature seed is of interest. Expression of adominant negative mutant of the present invention early in seeddevelopment can be desirable so as to increase cell divisions early inseed development. More cell divisions at this stage can lead to largerembryos. Embryo composition is approximately 33% oil therefore a largerembryo will lead to increases in oil content. A promoter suitable forexpression of the current invention in embryo and lower or no expressionin other plant tissues is of interest to increase seed oil content. Ofparticular interest are those promoter sequences that initiateexpression in early phase-specific embryo development. An earlyphase-specific promoter is a promoter that initiates expression of aprotein prior to day 7 after pollination (walking stick) in Arabidopsisor an equivalent stage in another plant species. Examples of promotersequences of particular interest include a promoter for the amino acidpermease gene (AAP1) (e.g., the AAP1 promoter from Arabidopsisthaliana), a promoter for the oleate 12-hydroxylase:desaturase gene(e.g., the promoter designated LFAH12 from Lesquerella fendleri), apromoter for the 2S2 albumin gene (e.g., the 2S2 promoter fromArabidopsis thaliana), a fatty acid elongase gene promoter (FAE1) (e.g.,the FAE1 promoter from Arabidopsis thaliana), and the leafy cotyledongene promoter (LEC2) (e.g., the LEC2 promoter from Arabidopsisthaliana). The AAP1, LFAH12, 2S2, and FAE1 promoters are inactive in theearliest stage of embryo development. They become transcriptionallyactive at progressively later stages in development starting with AAP1followed by LFAH12, 2S2, and then FAE. All four promoters then remainactive through later embryonic development stages. The LEC2 promoter hasan inverse expression profile. It is active in very early embryodevelopment and then its activity declines gradually through laterstages. Other embryo-specific promoters of interest include thepromoters from the following genes: Seedstick (Pinvopich et al., Nature424:85-88, 2003), Fbp7 and Fbp11 (Petunia Seedstick) (Colombo et al.,Plant Cell. 9:703-715, 1997), Banyuls (Devic, Plant J., 19:387-398,1999), ABI3 (Ng et al., Plant. Mol. Biol. 54:25-38, 2004), agl-15, Agl18(Lehti-Shiu et al., Plant Mol. Biol. 58:89-107, 2005), Phel (Kohler,Genes Develop. 17:1540-1553, 2003), emb175 (Cushing et al., Planta.221:424-436, 2005), L11 (Kwong et al., Plant Cell 15:5-18, 2003), Lec1(Lotan, Cell 93:1195-1205, 1998), Fusca3 (Kroj et al., Development130:6065-6073, 2003), TT12 (Debeaujon et al., Plant Cell 13:853-871,2001), TT16 (Nesi et al., Plant Cell 14:2463-2479, 2002), A-RZf (Zou andTaylor, Gene 196:291-295, 1997), TTG1 (Walker et al., Plant Cell11:1337-1350, 1999), TT1 (Sagasser et al., Genes Dev. 16:138-149, 2002),TT8 (Nesi et al., Plant Cell 12:1863-1878, 2000), and Gea-8 (carrot)(Lin et al., J. Exp. Botany 50:1139-1147, 1999) promoters. Embryospecific promoters from monocots include Globulin, Knox (rice)(Postma-Haarsma, Plant Mol. Biol. 39:257-271, 1999), Oleosin (Plant,Plant Mol. Biol. 25:193-205, 1994), Keddie, Plant Mol. Biol. 24:327-340,1994), Peroxiredoxin (Per1) (Haslekas et al., Plant Mol. Biol.36:833-845, 1998), Haslekas et al., Plant Mol. Biol. 53:313-326, 2003),HvGAMYB (Diaz et al., Plant J. 29:453-464, 2002) and SAD1(Isabel-LaMoneda et al., Plant J. 33:329-340, 1999) from Barley, and ZeaMaize Hybrid proline rich protein promoters (Jose-Estanyol et al., PlantCell 4:413-423, 1992; Jose-Estanyol et al., Gene 356:146-152, 2005).

Promoters of seed storage proteins are also of particular interest asseed storage proteins can represent up to 90% of total seed protein inmany plants. The seed storage proteins are strictly regulated, beingexpressed almost exclusively in seeds in a highly tissue-specific andstage-specific manner (Higgins et al., Ann. Rev. Plant Physiol.35:191-221, 1984; Goldberg et al., Cell 56:149-160, 1989). Moreover,different seed storage proteins may be expressed at different stages ofseed development. Expression of seed-specific genes has been studied ingreat detail (see reviews by Goldberg et al., supra, and Higgins et al.,supra). Examples of seed-specific promoters include LFAH12 ofArabidopsis and other plants, and the 5′ regulatory regions of anArabidopsis oleosin gene as described in United U.S. Pat. No. 5,977,436to Thomas et al. (incorporated in its entirety by reference), which whenoperably linked to either the coding sequence of a heterologous gene orsequence complementary to a native plant gene, direct expression of theheterologous gene or complementary sequence in a plant seed.

Suitable seed storage protein promoters for dicotyledonous plantsinclude, for example, bean β-phaseolin, lectin, and phytohemagglutininpromoters (Sengupta-Gopalan, et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324, 1985; Hoffman et al., Plant Mol. Biol. 11:717-729, 1988;Voelker et al., EMBO J. 6:3571-3577, 1987); rapeseed (Canola) napinpromoter (Radke et al., Theor. Appl. Genet. 75:685-694, 1988); soybeanglycinin and conglycinin promoters (Chen et al., EMBO J. 7:297-302,1988; Nielson et al., Plant Cell 1:313-328, 1989, Harada et al., PlantCell 1:415-425, 1989; Beachy et al., EMBO J. 4:3047-3053, 1985); soybeanlectin promoter (Okamuro et al., Proc. Natl. Acad. Sci. USA83:8240-8244, 1986); soybean Kunitz trypsin inhibitor promoter(Perez-Grau et al., Plant Cell 1:1095-1109, 1989; Jofuku et al., PlantCell 1:1079-1093, 1989); potato patatin promoter (Rocha-Sosa et al.,EMBO J. 8:23-29, 1989); pea convicilin, vicilin, and legumin promoters(Rerie et al., Mol. Gen. Genet. 259:148-157, 1991; Newbigin et al.,Planta 180:461-470, 1990; Higgins et al., Plant Mol. Biol. 11:683-695,1988; Shirsat et al., Mol. Gen. Genetics 215:326-331, 1989); and sweetpotato sporamin promoter (Hattori et al., Plant Mol. Biol. 14:595-604,1990).

For monocotyledonous plants, seed storage protein promoters useful inthe practice of the invention include, e.g., maize zein promoters(Schernthaner et al., EMBO J. 7:1249-1255, 1988; Hoffman et al., EMBO J.6:3213-3221, 1987 (maize 15 kD zein)); maize 18 kD oleosin promoter (Leeet al., Proc. Natl. Acad. Sci. USA 888:6181-6185, 1991); waxy promoter;shrunken-1 promoter; globulin 1 promoter; shrunken-2 promoter; riceglutelin promoter; barley hordein promoter (Marris et al., Plant Mol.Biol. 10:359-366, 1988); RP5 (Su et al., J. Plant Physiol. 158:247-254,2001); EBEL and 2 maize promoters (Magnard et al., Plant Mol. Biol.53:821-836, 2003) and wheat glutenin and gliadin promoters (U.S. Pat.No. 5,650,558; Colot et al., EMBO J. 6:3559-3564, 1987).

Also suitable for practice of the present invention are promoters ofgenes for B. napus isocitratelyase and malate synthase (Comai et al.,Plant Cell 1:293-300, 1989); delta-9 desaturase from safflower (Thompsonet al., Proc. Natl. Acad. Sci. USA 88:2578-2582, 1991) and castor(Shanklin et al., Proc. Natl. Acad. Sci. USA 88:2510-2514, 1991); acylcarrier protein (ACP) from Arabidopsis (Post-Beittenmiller et al., Nucl.Acids Res. 17:1777, 1989), B. napus (Safford et al., Eur. J. Biochem.174:287-295, 1988), and B. campestris (Rose et al., Nucl. Acids Res.15:7197, 1987); β-ketoacyl-ACP synthetase from barley (Siggaard-Andersenet al., Proc. Natl. Acad. Sci. USA 88:4114-4118, 1991); and oleosin fromZea mays (Lee et al., Proc. Natl. Acad. Sci. USA 88:6181-6185, 1991),soybean (Genbank Accession No. X60773) and B. napus (Lee et al., PlantPhysiol. 96:1395-1397, 1991).

Other promoters useful in the practice of the invention are known tothose of skill in the art. Moreover, known methods can be used toisolate additional promoters suitable for use in accordance with thepresent invention. For example, differential screening techniques can beused to isolate promoters expressed at specific (developmental) times,such as during fruit development.

Promoters of seed specific genes operably linked to heterologous codingsequences in chimeric gene constructs also maintain their temporal andspatial expression pattern in transgenic plants. Such examples includeuse of Arabidopsis thaliana 2S seed storage protein gene promoter toexpress enkephalin peptides in Arabidopsis and B. napus seeds(Vandekerckhove et al., Bio/Technology 7:929-932, 1989), bean lectin andbean β-phaseolin promoters to express luciferase (Riggs et al., PlantSci. 63:47-57, 1989), and wheat glutenin promoters to expresschloramphenicol acetyl transferase (Colot et al., supra).

Attaining the proper level of expression of the nucleic acid fragmentsof the invention may require the use of different chimeric genesutilizing different promoters. Such chimeric genes can be transferredinto host plants either together in a single expression vector orsequentially using more than one vector.

In addition, enhancers are often required or helpful to increaseexpression of the gene of the invention. It is necessary that theseelements be operably linked to the sequence that encodes the desiredproteins and that the regulatory elements are operable. Enhancers orenhancer-like elements may be either the native or chimeric nucleic acidfragments. This would include viral enhancers such as that found in the35S promoter (Odell et al., Plant Mol. Biol. 10:263-272, 1988),enhancers from the opine genes (Fromm et al, Plant Cell 1:977-984,1989), or enhancers from any other source that result in increasedtranscription when placed into a promoter operably linked to the nucleicacid fragment of the invention. For example, a construct may include theCaMV 35S promoter with dual transcriptional enhancer linked to theTobacco Etch Virus (TEV) 5′ nontranslated leader. The TEV leader acts asa translational enhancer to increase the amount of protein made.

Suitable promoter elements, such as those described herein, can be fusedto the mutant CKI nucleic acid sequences and a suitable terminator(polyadenylation region) according to well established procedure.

Transgenic Plants

In another aspect of the present invention, transgenic plants comprisinga mutant CKI transgene are provided. Transgenic plants expressing amutant CKI polypeptide can be obtained, for example, by transferring atransgenic vector (e.g., a plasmid or viral vector) encoding the mutantpolypeptide into a plant. Typically, when the vector is a plasmid, thevector also includes a selectable marker gene such as, e.g., theneomycin phosphotransferase II (nptII) gene encoding resistance tokanamycin. The most common method of plant transformation is performedby cloning a target transgene into a plant transformation vector that isthen transformed into Agrobacterium tumifaciens containing a helperTi-plasmid as described in Hoeckema et al. (Nature 303:179-181, 1983).The Agrobacterium cells containing the transgene vector are incubatedwith leaf slices of the plant to be transformed as described by An etal. (Plant Physiology 81:301-305, 1986). (See also Hooykaas, Plant Mol.Biol. 13:327-336, 1989). Transformation of cultured plant host cells isnormally accomplished through Agrobacterium tumifaciens, as describedabove. Cultures of host cells that do not have rigid cell membranebarriers are usually transformed using the calcium phosphate method asoriginally described by Graham et al. (Virology 52:546, 1978) andmodified as described in sections 16.32-16.37 of Sambrook et al., supra.However, other methods for introducing DNA into cells such as Polybrene(Kawai et al., Mol. Cell. Biol 4:1172, 1984), protoplast fusion(Schaffner, Proc. Natl. Acad. Sci. USA 77:2163, 1980), electroporation(Neumann et al., 1982 EMBO J. 1:841, 1982), and direct microinjectioninto nuclei (Capecchi, Cell 22:479, 1980) can also be used. Transformedplant calli can be selected through the selectable marker by growing thecells on a medium containing, e.g., kanamycin and appropriate amounts ofa phytohormone such as naphthalene acetic acid and benzyladenine forcallus and shoot induction. The plant cells can then be regenerated andthe resulting plants transferred to soil using techniques well known tothose skilled in the art.

In addition to the methods described above, a large number of methodsare known in the art for transferring cloned DNA into a wide variety ofplant species, including gymnosperms, angiosperms, monocots and dicots(see, e.g., Methods in Plant Molecular Biology (Glick and Thompson eds.,CRC Press, Boca Raton, Fla., 1993); Vasil, Plant Mol. Biol. 25:925-937,1994; and Komari et al., Current Opinions Plant Biol. 1:161-165, 1998(general reviews); Loopstra et al., Plant Mol. Biol. 15:1-9, 1990; andBrasileiro et al., Plant Mol. Biol. 17:441-452, 1991 (transformation oftrees); Eimert et al., Plant Mol. Biol. 19:485-490, 1992 (transformationof Brassica); Hiei et al., Plant J 6:271-282, 1994; Hiei et al., PlantMol. Biol. 35:205-218, 1997; Chan et al., Plant Mol. Biol. 22:491-506,1993; U.S. Pat. Nos. 5,516,668 and 5,824,857 (rice transformation); andU.S. Pat. No. 5,955,362 (wheat transformation); U.S. Pat. No. 5,969,213(monocot transformation); U.S. Pat. No. 5,780,798 (corn transformation);U.S. Pat. Nos. 5,959,179 and 5,914,451 (soybean transformation).Representative examples include electroporation-facilitated DNA uptakeby protoplasts (Rhodes et al., Science 240:204-207, 1988; Bates, MethodsMol. Biol. 111:359-366, 1999; D'Halluin et al., Methods Mol. Biol.111:367-373, 1999; U.S. Pat. No. 5,914,451); treatment of protoplastswith polyethylene glycol (Lyznik et al., Plant Molecular Biology13:151-161, 1989; Datta et al., Methods Mol. Biol., 111:335-334, 1999);and bombardment of cells with DNA laden microprojectiles (Klein et al.,Plant Physiol. 91:440-444, 1989; Boynton et al., Science 240:1534-1538,1988; Register et al., Plant Mol. Biol. 25:951-961, 1994; Barcelo etal., Plant J. 5:583-592, 1994; Vasil et al., Methods Mol. Biol.111:349-358, 1999; Christou, Plant Mol. Biol. 35:197-203, 1997; Finer etal., Curr. Top. Microbiol. Immunol. 240:59-80, 1999). Additionally,plant transformation strategies and techniques are reviewed in Birch,Ann Rev Plant Phys Plant Mol. Biol. 48:297, 1997; Forester et al., Exp.Agric. 33:15-33, 1997. Minor variations make these technologiesapplicable to a broad range of plant species.

In the case of monocot transformation, particle bombardment is typicallythe method of choice. However, monocots such as maize can also betransformed by using Agrobacterium transformation methods as describedin U.S. Pat. No. 5,591,616 to Hiei et al. Another method to effectmonocot transformation, e.g., corn, mixes cells from embryogenicsuspension cultures with a suspension of fibers (5% w/v, Silar SC-9whiskers) and plasmid DNA (1 μg/ul) and which is then placed eitherupright in a multiple sample head on a Vortex Genie II vortex mixer(Scientific Industries, Inc., Bohemia, N.Y., USA) or horizontally in theholder of a Mixomat dental amalgam mixer (Degussa Canada Ltd.,Burlington, Ontario, Canada). Transformation is then carried out bymixing at full speed for 60 seconds (for example with a Vortex Genie II)or shaking at fixed speed for 1 second (Mixomat). This process resultsin the production of cell populations out of which stable transformantscan be selected. Plants are regenerated from the stably transformedcalluses and these plants and their progeny can be shown by Southernhybridization analysis to be transgenic. The principal advantages of theapproach are its simplicity and low cost. Unlike particle bombardment,expensive equipment and supplies are not required. The use of whiskersfor the transformation of plant cells, particularly maize, is describedin, for example, U.S. Pat. No. 5,464,765 to Coffee et al.

U.S. Pat. No. 5,968,830 to Dan et al. describes methods of transformingand regenerating soybean. U.S. Pat. No. 5,969,215 to Hall et al.,describes transformation techniques for producing transformed Betavulgaris plants, such as the sugar beet.

Each of the above transformation techniques has advantages anddisadvantages. In each of the techniques, DNA from a plasmid isgenetically engineered such that it contains not only the gene ofinterest, but also selectable and screenable marker genes. A selectablemarker gene is used to select only those cells that have integratedcopies of the plasmid (the construction is such that the gene ofinterest and the selectable and screenable genes are transferred as aunit). The screenable gene provides another check for the successfulculturing of only those cells carrying the genes of interest.

Traditional Agrobacterium transformation with antibiotic resistanceselectable markers is problematical because of public opposition thatsuch plants pose an undue risk-of spreading anibiotic tolerance toanimals and humans. Such antibiotic markers can be eliminated fromplants by transforming plants using the Agrobacterium techniques similarto those described in U.S. Pat. No. 5,731,179 to Komari et al.Antibiotic resistance issues can also be effectively avoided by the useof bar or pat coding sequences, such as is described in U.S. Pat. No.5,712,135. These preferred marker DNAs encode second proteins orpolypeptides inhibiting or neutralizing the action of glutaminesynthetase inhibitor herbicides phosphinothricin (glufosinate) andglufosinate ammonium salt (Basta, Ignite).

The plasmid containing one or more of these genes is introduced intoeither plant protoplasts or callus cells by any of the previouslymentioned techniques. If the marker gene is a selectable gene, onlythose cells that have incorporated the DNA package survive underselection with the appropriate phytotoxic agent. Once the appropriatecells are identified and propagated, plants are regenerated. Progenyfrom the transformed plants must be tested to insure that the DNApackage has been successfully integrated into the plant genome.

There are numerous factors that influence the success of transformation.The design and construction of the exogenous gene construct and itsregulatory elements influence the integration of the exogenous sequenceinto the chromosomal DNA of the plant nucleus and the ability of thetransgene to be expressed by the cell. A suitable method for introducingthe exogenous gene construct into the plant cell nucleus in a non-lethalmanner is essential. Importantly, the type of cell into which theconstruct is introduced must, if whole plants are to be recovered, be ofa type which is amenable to regeneration, given an appropriateregeneration protocol.

Methods of Use

In accordance with another aspect of the present invention, celldivision in a plant cell is modulated, e.g., increased. Plant cellsamenable to modulation, such as an increase of cell division using themethods described herein, are plant cells that express a wild-type CKIpolypeptide having separate cyclin and CDK binding regions. Generally,the methods include expressing within the plant cell a mutant CKIpolypeptide as described herein, and allowing the mutant CKI polypeptideto inhibit wild-type CKI biological activity within the plant cell. Themutant CKI polypeptide is expressed from a recombinant nucleic acidencoding the mutant protein. Further, in certain embodiments, the methodfurther includes introducing into the plant cell the recombinant nucleicacid encoding the mutant CKI polypeptide. Recombinant nucleic acidsencoding mutant CKI polypeptides, including construction of suchrecombinant molecules and their introduction into plant cells, aredescribed generally supra, and are further exemplified in the Examples,infra. Modulation of cell division can be carried out in a plant celleither in vivo, such as described hereinabove with respect to transgenicplants. Alternatively, the method can be performed in a plant cell invitro.

As described hereinabove, the mutant CKI polypeptide includes a CKIamino acid sequence having at least one modification relative to areference CKI polypeptide. With respect to the method for modulation ofcell division, the reference CKI polypeptide can be either the wild-typeplant CKI polypeptide expressed within the plant cell in whichmodulation of cell division is to be carried out (typically theendogenous wild-type CKI). Alternatively, because use of a mutant CKIprotein as described herein can allow for dominant negative antagonistactivity against structural homologues or family members of thewild-type CKI from which the mutant is derived, the reference CKIpolypeptide can be a wild-type CKI polypeptide heterologous to thewild-type CKI polypeptide expressed with the plant cell and which iscapable of providing substantially equivalent wild-type function. Thus,in some cases, a mutant CKI protein, not derived from the endogenous CKIprotein, is expressed within a plant cell to inhibit endogenouswild-type CKI function (e.g., a mutant CKI polypeptide derived from anArabidopsis thaliana wild-type KRP protein can be used to inhibitwild-type KRP function in non-heterologous plant cells such as, forexample, Brassica napus, Glycine max, Maize cells and the like). Otherheterologous mutant CKI polypeptides derived from other plants, such asBrassica napus, and the like, can also be used.

Further, in certain embodiments of the method, a plurality of relatedCKIs are simultaneously inhibited within a plant cell using a mutant CKIof the present invention. For example, in some embodiments, allendogenous CKIs within a family (e.g., KRP family members) aresimultaneously inhibited. As noted above, a mutant CKI protein asdescribed herein allows for dominant negative antagonist activity withinstructural homologues or family members of the wild-type CKI from whichthe variant is derived, thereby allowing for such simultaneousinhibition of multiple homologues or family members. Thus, in typicalembodiments, a plurality of (and preferably all) CKIs having substantialsequence identity or similarity within the targeted CDK or cyclinbinding region are inhibited within a plant cell. For example, incertain embodiments, as KRP family members share substantial sequenceidentity within the CDK binding region, which is primarily responsiblefor inhibition of cyclin-CDK kinase activity, a plurality of, andpreferably all, endogenous KRP family members within a plant cell areinhibited via expression of a mutant KRP CKI so as to modulate celldivision.

As described above, by effectively blocking endogenous CKI proteins frominhibiting cyclin/CDK activities, cell division within a plant cell ismodulated. Such modulation includes acceleration of progression throughthe cell cycle and, ultimately, increased cellular proliferation.Accordingly, using the methods set forth herein in vivo (e.g.,transgenic plants, see supra), an increase in crop yields and/or seedsize, increased plant vigor, increased root mass, increased fruit size,and the like can be achieved.

Increased crop yield can manifest itself in a variety of forms dependingon the specific plant tissue and plant species in which the plant cellcycle has been modulated by blocking endogenous CKI proteins frominhibiting cyclin/CDK activities.

In seed crops such as corn, rice, wheat, barley, soybean, canola,increased yield can take the form of increased total seed number,increased seed size, or both increased seed number and seed size. In oneembodiment, increased yield is obtained in transgenic varieties of anyof the seed crops by expressing a transgene encoding a mutant CKIprotein resulting in increased cell division and increased total seednumber, larger seeds, or both increased seed number and seed size. Inone embodiment, expression of the mutant CKI protein transgene iscontrolled by a constitutive promoter. In another exemplary embodiment,expression of the mutant CKI protein transgene is controlled by aseed-specific promoter targeting the effect of the mutant CKI protein tothe seed which is the agronomically important component of a seed crop.

For oilseed crops such as soybean, canola, camelina, flax, corn,safflower, or sunflower, and the like, where the desired product is oil,increased yield takes the form of greater oil yield per plant. Oil isderived from the embryo in the seed. In one embodiment, increased oilyield is obtained in transgenic varieties of any of the oilseed crops byexpressing a transgene encoding a mutant CKI protein causing increasedcell division leading to increased total seed number, increased embryosize, or both increased seed number and embryo size. Increased totalseed number per plant results in increased total oil yield per plant.Increased embryo size results in greater oil content per seed andincreased total oil yield per plant. In a preferred embodiment,expression of the mutant CKI protein transgene is controlled by anembryo-specific promoter giving increased cell division in early embryodevelopment resulting in a larger embryo, an increased oil quantity perseed, and a corresponding increase in total oil yield per plant.

Non-seed biomass production is the main yield component of crops such asalfalfa, lettuce, tobacco, eucalyptus, and poplar. Increased yield insuch crops would be seen in increased overall growth of the plantresulting in enhanced accumulation of biomass. In one embodiment,increased biomass is obtained in transgenic varieties of any of thebiomass crops by expressing a transgene encoding a mutant CKI proteincausing increased cell division leading to increased growth and biomass.In one embodiment, expression of the mutant CKI protein transgene iscontrolled by a promoter giving constitutive expression of the mutantCKI protein encoding transgene in most or all the tissues of the plant.In a preferred embodiment, expression of the mutant CKI proteintransgene is controlled by a tissue-specific promoter targetingexpression of the transgene to the agronomically important component ofthe plant such as the leaf in lettuce or tobacco or the trunk ineucalyptus or poplar to increase biomass accumulation in the targettissue.

Sugar or cellulose is the primary yield component of crops grown forproduction of ethanol including sugarcane, sugar beet, corn, andswitchgrass. In one embodiment, increased sugar is obtained intransgenic varieties of any of the sugar producing crops such assugarcane or sugar beet by expressing a transgene encoding a mutant CKIprotein causing increased cell division, increased growth of the sugaraccumulating tissue of the plant, and increased total sugar content perplant. In one embodiment, expression of the mutant CKI protein transgenein sugar producing crops is controlled by a promoter giving constitutiveexpression of the mutant CKI protein encoding transgene in most or allthe tissues of the plant. In a preferred embodiment, expression of themutant CKI protein transgene is controlled by a tissue-specific promotertargeting expression of the transgene to the sugar accumulating tissueof the plant such as the cane in sugarcane or the root in sugar beetthereby increasing cell proliferation and growth of the sugar storageand accumulation tissue of the plant resulting in increased total sugarcontent. In another embodiment, increased cellulose is obtained intransgenic varieties of any of the cellulose producing crops such ascorn or switchgrass by expressing a transgene encoding a mutant CKIprotein causing increased cell division and cell wall synthesis therebyincreasing the content of cellulose which is a component of the cellwall. In one embodiment, expression of the mutant CKI protein transgeneis controlled by a promoter giving constitutive expression of the mutantCKI protein encoding transgene in most or all the tissues of the plant.The consequent general increase in cell proliferation results inincreased cell wall deposition and therefore increased total cellulosecontent of the plant.

EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1 Production/Purification of an Active Cyclin:CDK Complex andthe Production/Purification of Krp Molecules

Insect Cells and Media

The baculovirus expression system is a versatile eukaryotic system forheterologous gene expression. This system provides correct proteinfolding, disulfide bond formation and other important post-translationalmodifications. All methods were taken from the Baculovirus expressionvector system: Procedures and methods manual. (BD Biosciences,Pharmingen, San Diego, Calif. 6th ed.). Sf9 insect cells were grown at27° C. in TNM-FH insect cell media (BD Biosciences) for the reportedstudies. It should be noted that alternative media are well known to theskilled artisan and are also useful. Similarly, alternative insect celllines such as Sf21 and High Five™ cells will also work for virusproduction and protein production.

Western Blot and Immunoprecipitations.

The recombinant protein expressed in insect cells was monitored byWestern blot. Protein extracts (35 μg) were boiled in the presence ofLaemmli buffer, run on 10% or 12% SDS-PAGE gels and transferred to aPVDF membrane using a submerged transfer apparatus (BioRad). Followingthe transfer, the membrane was blocked in TBS-T (25 mM Tris pH 7.5; 75mM NaCl; 0.05% Tween) containing 5% non-fat dry milk powder. Primaryantibody was used at 1:1000 dilution overnight in TBS-T blocking buffer.Blots were washed three times 15 minutes at room temperature. Anappropriate secondary antibody conjugated to horse radish peroxidase(HRP) was used at 1:10,000 dilution in TBS-T blocking buffer. Blots wereincubated in secondary antibody for 1 hour and then washed three timesin TBS-T, 15 min. each. Blots were then processed as described in theECL system protocol (Amersham Biosciences). Antibodies commonly usedwere: anti-flag M2 monoclonal antibody (Sigma), anti-HA monoclonal orpolyclonal antibody (Babco), anti-PSTAIR antibody (Sigma-Aldrich),anti-myc monoclonal or polyclonal (A-14) (Santa Cruz Biotechnology).Secondary antibodies used were anti-mouse IG-HRP, and anti-rabbit IG-HRP(GE Healthcare).

Immunoprecipitations were routinely performed to monitor complexformation between AtCyclinD2;1, AtCDKA and Krp molecules. Proteinextracts (14 μg) were diluted in 0.5 ml binding buffer (100 mM SodiumPhosphate buffer pH 7.0, 150 mM NaCl, 1% Triton 100 plus proteaseinhibitors). Protein/antibody mixture was rocked gently at 4° C. forabout two hours and then the appropriate antibody was added. (2 μg ofanti-flag M2 antibody; 5 μl of anti-HA antibody; 5 μl of anti-mycpolyclonal). Protein A sepharose was added to give a 10 μl bed volume.Immunoprecipitates were gently mixed for 1 hour at 4° C. and then washed3 times with 1 ml of binding buffer. Protein complexes bound to theProtein-A sepharose beads were then boiled in the presence of Laemmlibuffer. Protein complexes were resolved on either 10% or 12% SDS-PAGEgels and transferred to PVDF membrane. Membranes were blotted asdescribed above.

Baculovirus Vector Construction

Arabidopsis cyclin D2;1 (AtcyclinD2;1) and Arabidopsis CDKA (AtCDKA)cDNA sequences were epitope tagged and cloned into a baculovirustransfer vector (BD Biosciences). Other transfer vector systems known tothe artisan can also be used. AtCyclinD2;1 was tagged with the FLAGepitope (Sigma-Aldrich) on the N-terminus by adding Met Asp Tyr Lys AlaPhe Asp Asn Leu (MDYKAFDNL) amino acid sequence (SEQ ID No:18) by PCRand then cloned into the pAcHLT transfer vector (BD Biosciences). OtherBaculovirus transfer vector systems such as Baculodirect (Invitrogen),can also be used for this purpose. The hemagglutinin (HA) epitope aminoacid sequence Tyr Pro Tyr Asp Val Pro Asp Tyr Ala (YPYDVPDYA; SEQ IDNO:19) was placed in frame with the 5′ end of AtCDKA by PCR and thencloned into the pVL1393 transfer vector (AB Vector, CA). The cyclin andCDK were epitope tagged to enable identification by Western blot and forimmunoprecipitation experiments. The cyclin and or the CDK can also beused lacking the tags. Other compatible transfer vector systems can alsobe used.

Recombinant Virus Production

The Baculovirus genome used is Baculogold Bright Baculovirus (BDBiosciences). Alternative Baculovirus genomes can also be used. The Flagtagged version of AtcyclinD2;1 was introduced into a nonessential regionthe baculovirus viral genome using homologous recombination. Homologousrecombination occurred when the transfer vector containing cyclin D2;1was co-transfected with the linearized BD Baculogold Bright BaculovirusDNA into Sf9 insect cells. Sf9 cells were seeded at 2×10⁶ cells on 60 mmdish and transiently co-transfected with 2 μg cyclin D2;1 transfervector (pAcHLT-cyclinD2;1) plus 0.5 μg linearized BD Baculogold BrightBaculovirus DNA using Fugene 6 transfection reagent according tomanufacturer's protocol (Roche Diagnostics). After 4 hours oftransfection the Fugene/DNA solution was removed and replaced with 3 mlof TNM-FH media. Four (4) days later, the supernatant was collected andsubsequently used to infect more cells for amplification of the virus.This amplification was repeated until the virus titer was at least 10⁹virus particles/ml. The virus was amplified by infecting Sf9 cells at amultiplicity of infection (moi) of <1. The virus titer was monitoredusing light and fluorescence microscopy.

The HA tagged version of AtCDKA 1 was introduced into a nonessentialregion the baculovirus viral genome using homologous recombination.Homologous recombination occurred when the transfer vector containingcyclin D2;1 was cotransfected with the linearized BD Baculogold BrightBaculovirus DNA into Sf9 insect cells. Sf9 cells were seeded at 2×10⁶cells on 60 mm dish and transiently co-transfected with 2 μg AtCDKAtransfer vector (pVL1393-AtCDKA) plus 0.5 μg linearized BD BaculogoldBright Baculovirus DNA using Fugene 6 transfection reagent according tothe manufacturer's protocol (Roche Diagnostics). After 4 hours oftransfection the Fugene/DNA solution was removed and replaced with 3 mlof TNM-FH media. Four (4) days later, the supernatant was collected andsubsequently used to infect more cells for virus amplification. Thisamplification was repeated until the virus titer was at least 10⁹ virusparticles/ml. The virus was amplified by infecting Sf9 cells at amultiplicity of infection (MOI) of <1. The virus titer was monitoredusing light and fluorescence microscopy.

Recombinant Protein Production in Insect Cells.

Production of Flag tagged AtcyclinD2;1 protein: Tagged AtcyclinD2;1 wasachieved by infecting S. frugiperda Sf9 cells with AtcyclinD2;1baculovirus. To this end, Sf9 cells were grown in suspension at 2×10⁶/mlwere infected with recombinant baculovirus at an MOI>5 (but other higheror slightly lower MOIs will also work) for about 4 to 5 days and thenharvested. Cells were collected and centrifuged at 3000 rpm at 4° C. Thecell pellet was washed with fresh media and then centrifuged at 3000 rpmat 4° C. The pellet was frozen at −80° C. or immediately lysed. Lysisbuffer consisted of 20 mM Hepes pH 7.5, 20 mM NaCl, 1 mM EDTA, 20%glycerol, 20 mM MgCl₂ plus protease inhibitors (Complete Mini, EDTAfree, Boehringer Mannheim), 1 tablet per 10 ml lysis buffer. The celllysate was sonicated on ice 2 times for 15 seconds. Protein lysate wasthen centrifuged at 40,000 rpm in a Beckman TLA 100.2 rotor for 2 hours.The supernatant containing the tagged AtCyclinD2;1 was aliquoted andfrozen at −20° C. Expression was monitored by Western blot usinganti-Flag M2 monoclonal antibody (Sigma-Aldrich).

Production of tagged AtCDKA was achieved by infection of S. frugiperdaSf9 cells with AtCDKA baculovirus and processed in the same manner asdescribed above. Expression was monitored by Western blot using anti-HAmonoclonal or polyclonal antibody (Babco). Expression can also bemonitored by Western blot using anti-PSTAIR antibody (Sigma-Aldrich).

An active kinase complex of AtcyclinD2;1/AtCDKA was prepared byco-infecting of S. frugiperda Sf9 cells with AtcyclinD2;1 (MOI>5) plusAtCDKA (MOI>5) baculovirus. The active complex was purified as describedabove. Protein expression was monitored by Western blot of insect cellextracts using anti-Flag M2 antibody or anti-HA antibody. Theinteraction of AtcyclinD2;1 and AtCDKA was monitored byco-immunoprecipitation as described infra.

Kinase Assay

An in vitro assay was developed to test various KRP/ICK moleculesability to inhibit cyclin/CDK complexes.

Kinase activity in protein extracts from insect cells infected withindividual baculovirus or a co-infection with the two baculovirus wasmonitored with a standard kinase assay. Histone HI (HHI) was theprinciple substrate used but recombinant tobacco retinoblastoma protein(Nt Rb) could also be used as the substrate (see Koroleva et al., PlantCell 16, 2346-79, 2004). Kinase assays were performed as follows: 7 μgof insect cell protein extract was added to a kinase buffer cocktail(KAB: 50 mM Tris pH 8.0, 10 mM MgCl₂, 10 μM ATP plus 0.5 μCi/ml ³²PγATPand 2 μg of HHI) to a final volume of 30 μl. The reactions wereincubated at 27° C. for 30 minutes. The kinase reaction was stopped withan equal volume (30 μl) of 2× Laemmli buffer. [³²P] phosphateincorporation was monitored by autoradiography and/or Molecular DynamicsPhosphorImager following SDS-PAGE on 12% gels. Alternative bufferconditions for performing CDK kinase assays can also be used. (See,e.g., Wang and Fowke, Nature 386:451-452, 1997; Azzi et al., Eur. J.Biochem. 203:353-360, 1992; Firpo et al., Mol. Cell. Biol 14:4889-4901,1994.)

Results: Protein extract from insect cells infected with AtcyclinD2;1alone or AtCDKA alone showed no kinase activity using HHI as thesubstrate. Insect cells co-infected with AtcyclinD2;1 virus and AtCDKAvirus contained a robust kinase activity. Active CDK-like (cdc2-like)kinases can also be purified from plant protein tissue extracts or fromplant tissue culture cell extracts by using p13suc1 agarose beads (SeeWang and Fowke, Nature 386:451-452, 1997; Azzi et al., Eur. J. Biochem.203:353-360, 1992) and used in a similar assay described above and incompetition experiments described below in Examples 2 through 8.

Cloning of Krp cDNAs into Bacterial Expression Vector.

ATKrp1 Cloning

AtKrp1 cDNA was cloned from Arabidopsis cDNA by PCR using the followingoligonucleotides: (start) 5′ ATGGTGAGAAAATATAGAAAAGCT-3′; (SEQ ID NO:20) (end) 5′-TCACTCTAACTTTACCCATTCGTA-3′. (SEQ ID NO: 21)The resulting PCR fragment was subcloned into pCRII-TOPO vector(Invitrogen). The resulting vector AtKrp1#359 was sequenced to verifycorrect sequence to GenBank# U94772.5′ RACE of Brassica napus Krp1 Cloning

Blastn was used at the National Center for Biotechnology Informationwebsite to find Brassica sequences homologous to AtKRP1 cds. The searchyielded two EST candidates, CD820320 and CD829052, which turned out tobe identical sequences. CD820320 was taken through Jorja's blastx toverify that the translated nucleotide sequence of the EST significantlymatched the At KRP1 protein sequence.

CD829052 is 646 bp and includes the last 106 amino acids of a Brassicaoleracea KRP1 sequence and 321 bp of the 3′ UTR. A 5′ RACE primer(GSP1brasskrp1_(—)5′RACE:5′-CTCTGATAATTTAACCCACTCGTAGCGTCCTTCTAATGGCTTCTC-3′; SEQ ID NO:22) wasdesigned to retrieve a full-length Bn KRP1. The RACE primer containedthe last 45 nucleotides of the coding sequence of CD820320.

5′ RACE-ready cDNA was made from Brassica napus DH12075 leaf using theSMART RACE cDNA Amplification Kit (Clontech). 5′ RACE was performed onthis cDNA according to the kit instructions, except that pfu enzyme andbuffer (Stratagene) were used instead of Klentaq. The PCR conditionswere: an initial denaturation at 94° C. for 5 min, followed by 35 cyclesof 94° C. for 5 sec, 68° C. for 10 sec, 72° C. for 3 min. The resultingPCR product was a very faint band of approximately 600 bp. 2.5 μl ofthis PCR product was then re-amplified using the above PCR conditions.The resulting PCR product was cloned into TOPO Blunt vector (Invitrogen)and transformed into alpha gold cells (Bioline). Plasmid DNA from twotransformants were recovered and the inserts sequenced with M13 forwardand reverse primers. The sequence of one candidate insert was identicalto CD820320 and was designated Bn KRP1-II (no additional new sequencescame from the RACE). The sequence of the other candidate insert,designated Bn KRP1-I, was 94% identical to Bn KRP1-II at the nucleotidelevel in the coding region, and 86% identical at the amino acid levelfor the last 106 residues. The RACE retrieved the full coding sequenceof Bn KRP1-I with an additional 108 bp of 5′ UTR in TOPO Blunt vector(pTG313). The BnKrp1 coding sequence (SEQ ID NO:23) is shown below: 1atggtgagaaaatgcagaaaaactaaagggacggtgggagcttcgtctacgtatatgcagcttcgcagccggagaatcgt80 81ttacagatcggaaaaagctagctcgtcgtcgtcgtcttgttgcgcgagtaacaacaatggagttatagatcttgaggagg160 161aaagagatggtgagactgaaacgtcgtcgtgtcgacggagtagtaagaggaagctatttgaaaaccttagagaaaaagaa240 241tctatggagaattcacagcaaatcgtagctggttttgattccgccgtgaaagaatcatcggattgttgttgcagccggag320 321aacatctttgtcaacgacggaggagaaggggaaatcagcgacggagcaaccaccaacggcagtggagattgaagattttt400 401tcgtggaagctgagaaacagctccatgataatttcaagaagaagtataactttgatttcgaaaaggagaagccattagaa480 481 ggacgctacgagtgggttaaattatcagagtaa 513

The pET16b bacterial expression vector contains the sequence encoding 6Histidines (6×His, or (His)₆ or hexaHis) in a row to enable proteinpurification by immobilized metal affinity chromatography (Novagen).This vector was modified in such a way to also include a poly-Mycepitope immediately downstream and in frame with the 6×His codingsequence (pET16b-5MYC). Full length AtKrp1 cDNA was amplified from pTG#359 TopoIIAtKrp1 cds using two oligonucleotides that flank the entirecoding sequence. These oligonucleotides contained restriction enzymesites that facilitate cloning into the pET16B-5MYC vector (5′AtKrp1 BamHI/NdeI: ACGGATCCCATATGGTGAGA AAATATAG (SEQ ID NO:24) and 3′AtKrp1Xho I:ATCGCTCGAGTCACTCTAACTTTAC (SEQ ID NO:25). The resulting PCR fragment wassubcloned into the BamHI and XhoI site of pET16b-5myc. The resultingvector AtKrp #385 contained the AtKrp1 wild-type cDNA in frame with the6×His and myc tags.

The following two oligonucleotides were used to amplify the BnKrp1 cDNAthat adds a 5′ BamHI/NdeI site and XhoI restriction site on the 3′ endof BnKrp1 (5′ BnKrp1 BamHI/NdeI: ACGGATCCCATATGGTGAGAAAATGC (SEQ IDNO:26) and 3′ BnKrp1 XhoI: ATCGCTCGAGTCACTCTGATAATTTAAC (SEQ ID NO:27).The PCR fragment was amplified from pTG #313 (TopoII BnKrp1) andsubcloned into the BamHI and XhoI site of pET16b-5myc and sequenced. Theresulting vector BnKrp #461 contained the BnKrp1 wild-type cDNA in framewith the 6×His and myc tags.

Recombinant Krp Protein Expression in Bacteria and Purification.

All bacterial expression plasmids pET16b and pET16b-5MYC carryinginserts were transformed into BL21 Star (DE3) (Invitrogen). Bacterialcolonies from this fresh transformation was used to inoculate 400 ml ofLB containing 100 μg/ml of ampicillin and grown at 37° C. When theculture reached an OD₆₀₀ between 0.6 and 0.8 recombinant proteinexpression was induced with 0.5 mM isopropyl-D-thiogalactopyranoside(IPTG). Cells were then grown at 30° C. for three hours. Cells werecollected by centrifugation in a JLA 10.500 Beckman rotor. Bacterialcell pellet was either stored at −80° C. or lysed immediately. Bacteriawere lysed in 10 ml Phosphate lysis buffer (100 mM Phosphate buffer pH7.0, 150 mM NaCl, 1% Triton X100] containing protease inhibitors andlacking EDTA. The resuspended bacterial culture was sonicated 4×20seconds on wet ice at 40% power. Lysed cells were centrifuged at 14,000rpm in Beckman JA20.1 rotor for 15 minutes at 4° C. Cell pellet waswashed with 10 mls of Phosphate lysis buffer and the cell pellet wasagain collected by centrifugation. Tagged KRP molecules were mainlyinsoluble. Insoluble tagged KRP's were solubilized in Urea buffer (8MUrea, 100 mM Tris pH 7.5). The resuspended cell pellet was brieflysonicated 3×15 seconds on wet ice at 40% power. Urea-insoluble proteinswere eliminated by centrifugation at 14,000 rpm in Beckman JA20.1 rotorfor 15 minutes at 4° C. Tagged KRPs were purified in batch using BDTalon Co²⁺ metal affinity resin equilibrated in Urea buffer. Batchpurification was incubated at 4° C. 3 hrs to overnight under slowrotation. Slurry was loaded on a column and resin was washed with 36 bedvolumes of Urea buffer followed by a 12 bed volumes of Urea buffercontaining 5 mM Imidazole pH 7.5. Bound tagged KRP protein was elutedusing Urea buffer containing 300 mM Imidazole pH 7.5. Fractions weremonitored for tagged KRP by SDS-Page and/or by Bradford protein assay(BioRad). Refolding of the denatured tagged KRP1 was carried out usingstep-wise dilution dialysis. Fractions containing the majority of taggedKRP protein were combined and dialyzed in a 1M Urea, 100 mM Tris pH7.5,150 mM NaCl and 5 mM β-mercaptoethanol plus 5 mM benzamidine for 20 hrsat 4° C. Dialysis buffer was then changed to 0.5 M Urea, 100 mM Tris pH7.5, 150 mM NaCl and 5 mM β-mercaptoethanol plus 5 mM benzamidine andcontinued for an additional 12 hrs. Recombinant protein was collected,quantified by Bradford assay and stored at 4° C.

Mutagenesis of Krps.

The wild-type versions of AtKRP family members and BnKRP family memberswere subcloned into the basic pCRII-TOPO vector (Invitrogen) forsequencing and mutagenesis purposes.

The same AtKRP1 wild-type cDNA PCR fragment containing the engineered5′-BamHI site and the 3′ XhoI site (see “Cloning of Krp cDNAs intobacterial expression vector,” supra) was subcloned into pCRII-TOPOvector (Invitrogen) for mutagenesis. The resulting vector Topo-AtKrp#385B contained the AtKRP1 wild-type cDNA and was verified for correctsequence using standard automated sequencing. The same BnKRP1 wild-typecDNA fragment from above was subcloned into pCRII-TOPO vector(Invitrogen) for mutagenesis. The resulting vector Topo-BnKrp #539contained the BnKRP1 wild-type cDNA and was verified for correctsequence using standard automated sequencing analysis.

Site directed mutagenesis was performed according to the protocol forStratagene's QuikChange site-directed mutagenesis kit.

To construct BnKrp1# 462 with multiple amino acids substitutions in theputative cyclin binding region (E129A, E130A, I131A, D132A) the senseBnKrp1DbleMut#1 (GGA GCAACCACCAACGGCAGTGGCTGCTGCTGCTTTTTTCGTG; SEQ IDNO:28) and anti-sense BnKrp1DbleMut#1b(CACGAAAAAAGCAGCAGCAGCCACTGCCGTTGGTGGT TGCTCC; SEQ ID NO:29) were usedfor QuikChange site-directed mutagenesis with TopoBnKrp#539 as thetemplate. The mutagenesis product was sequenced to verify presence ofdesired mutations. The mutant product was then subcloned into theBamHI/XhoI site of pET16b-5MYC to ultimately yield BnKrp1#462.

To construct BnKrp1# 512 with two amino acids substitutions in thehighly conserved residues of the CDK binding region (F151A, F1153A) thesense BnKrp1 DbleMut#2 oligonucleotide(CCTTCTAATGGCTTCTCCTTTTCAGCATCAGCGTTATACTTCTTCT TGAA; SEQ ID NO:30) andanti-sense BnKrp1DbleMut#2b oligonucleotide (TTCAAGAAGAAGTATAACGCTGATGCTGAAAAGGAGAAGCCATTAGAAGG; SEQ ID NO:31) were used forQuikChange site-directed mutagenesis with TopoBnKrp#539 as the template.The mutagenesis product was sequenced to verify presence of desiredmutations and then subcloned into the BamHI/XhoI site of pET16b-5MYC.The same amino acid substitutions were also introduced into AtKRP1 usingAtKrp1-Topo # 385B as the template using the following oligonucleotidesQC ICK1 cds F173A, F175A-coding” 5′-TTCAAGAAGAAGTACAATGCCGATGCCGAGAAGGAGAAGCCATTA-3′; (SEQ ID NO:32) and “QC ICK1cdsF173A, F175A-noncod” 5′-TTATGGCTTCTCCTTCTGGGCATCGGCATTGTACTTCTTCTTGAA-3′ (SEQ ID NO:33).

For BnKrp1#463 with amino acids substitutions of the putative cyclinbinding region and highly conserved residues in the CDK binding region(E129A, E130A, I131A, D132A, +F151A, F153A) the sense BnKrp1DbleMut#2oligonucleotide ((CCTTCTAAT GGCTTCTCCTTTTCAGCATCAGCGTTATACTTCTTCTTGAA;SEQ ID NO:30) anti-sense BnKrp1DbleMut#2b oligonucleotide(TTCAAGAAGAAGTATAACGCTGATGCTG AAAAGGAGAAGCCATTAGAAGG; SEQ ID NO:32) wereused for QuikChange site-directed mutagenesis with BnKrp#462 as thetemplate. The mutagenesis product was sequenced to verify presence ofdesired mutations and then subcloned into the BamHI/XhoI site ofpET16b-5MYC.

To construct BnKrp1# 586 with a single amino acids substitution in theCDK binding region (K148A) the sense BnKrp1K148A oligonucleotide(GATAATTTCAAGAAG GCGTATAACTTTGATTTC; SEQ ID NO:34) and anti-senseBnKrp1K148A oligonucleotide (GAAATCAAAGTTATACGCCTTCTTGAAATTATC; SEQ IDNO:35) were used for QuikChange site-directed mutagenesis withTopoBnKrp#539 as the template. The mutagenesis product was sequenced toverify presence of desired mutations and then subcloned into theBamHI/XhoI site of pET16b-5MYC.

To construct BnKrp1# 587 with a single amino acids substitution in theCDK binding region (Y149A) the sense BnKrp1Y149A oligonucleotide(AATTTCAAGAAGAAG GCTAACTTTGATTTCGAA; SEQ ID NO:36) and anti-senseBnKrp1Y149A oligonucleotide (TTCGAAATCAAAGTTAGCCTTCTTCTTGAAATT; SEQ IDNO:37) were used for QuikChange site-directed mutagenesis withTopoBnKrp#539 as the template. The mutagenesis product was sequenced toverify presence of desired mutations and then subcloned into theBamHI/XhoI site of pET16b-5MYC.

To construct BnKrp1# 588 with a single amino acids substitution in theCDK binding region (N150A) the sense BnKrp1N150A oligonucleotide(TTCAAGAAGAAGTAT GCCTTTGATTTCGAAAAG; SEQ ID NO:38) and anti-senseBnKrp1N150A oligonucleotide (CTTTTCGAAATCAAAGGCATACTTCTTCTTGAA; SEQ IDNO:39) were used for QuikChange site-directed mutagenesis withTopoBnKrp#539 as the template. The mutagenesis product was sequenced toverify presence of desired mutations and then subcloned into theBamHI/XhoI site of pET16b-5MYC.

To construct BnKrp1# 572 with a single amino acids substitution in theCDK binding region (F151A) the sense BnKrp1F151A oligonucleotide(AGAAGAAGTATAACGCTGATTTCGGAAAGGA; SEQ ID NO:40) and anti-senseBnKrp1F151A oligonucleotide (TCCTTTTCGAAATCAGCGTTATACTTCTTCT; SEQ IDNO:41) were used for QuikChange site-directed mutagenesis withTopoBnKrp#539 as the template. The mutagenesis product was sequenced toverify presence of desired mutations and then subcloned into theBamHI/XhoI site of pET16b-5MYC.

To construct BnKrp1# 573 with a single amino acids substitution in theCDK binding region (F153A) the sense BnKrp1F153A oligonucleotide(AGTATAACTTTGATGCCGAAAAGGAGAAGCC; SEQ ID NO:42) and anti-senseBnKrp1F153A oligonucleotide (GGCTTCTCCTTTTCGGCATCAAAGTTATACT; SEQ IDNO:43) were used for QuikChange site-directed mutagenesis withTopoBnKrp#539 as the template. The mutagenesis product was sequenced toverify presence of desired mutations and then subcloned into theBamHI/XhoI site of pET16b-5MYC.

To construct BnKrp1# 553 with two amino acids substitutions in the CDKbinding region (K157A; P158A) the sense BnKrp1KP→AA oligonucleotide(GATTTCGAAAAGGA GGCGGCATTAGAAGGACGCT; SEQ ID NO:44) and anti-senseBnKrp1KP→AA oligonucleotide (AGCGTCCTTCTAATGCCGCCTCCTTTTCGAAATC)(SEQ IDNO:45) were used for QuikChange site-directed mutagenesis withTopoBnKrp#539 as the template. The mutagenesis product was sequenced toverify presence of desired mutations and then subcloned into theBamHI/XhoI site of pET16b-5MYC.

To construct BnKrp1# 554 with two amino acids substitutions in the CDKbinding region (R162A; Y163A) the sense BnKrp1RY→AA oligonucleotide(GCCATTAGAAGGA GCCGCCGAGTGGGTTAAATT; SEQ ID NO:46) and anti-senseBnKrp1RY→AA oligonucleotide (AATTTAACCCACTCGGCGGCTCCTTCTAATGGC; SEQ IDNO:47) were used for QuikChange site-directed mutagenesis withTopoBnKrp#539 as the template. The mutagenesis product was sequenced toverify presence of desired mutations and then subcloned into theBamHI/XhoI site of pET16b-5MYC.

To construct BnKrp1# 555 with two amino acids substitutions in the CDKbinding region (E164A; W165A) the sense BnKrp1EW→AA oligonucleotide(AGAAGGACGCTAC GCGGCGGTTAAATTATCAGA; SEQ ID NO: 48) and anti-senseBnKrp1EW→AA oligonucleotide (TCTGATAATTTAACCGCCGCGTAGCGTCCTTCT; SEQ IDNO: 49) were used for QuikChange site-directed mutagenesis withTopoBnKrp#539 as the template. The mutagenesis product was sequenced toverify presence of desired mutations and then subcloned into theBamHI/XhoI site of pET16b-5MYC.

To construct BnKrp1# 556 with two amino acids substitutions in the CDKbinding region K167A; L168A) the sense BnKrp1KL→AA oligonucleotide(CGCTACGAGTGGGT TGCAGCATCAGAGTGAGAGC; SEQ ID NO: 50) and anti-senseBnKrp1KL→AA oligonucleotide (GCTCTCACTCTGATGCTGCAACCCACTCGTAGCG; SEQ IDNO:51) were used for QuikChange site-directed mutagenesis withTopoBnKrp#539 as the template. The mutagenesis product was sequenced toverify presence of desired mutations and then subcloned into theBamHI/XhoI site of pET16b-5MYC.

To construct BnKrp1# 574 with multiple amino acid substitutions in theCDK binding region (F151A; F153A; E164A; W165A) the BnKrp1F151A, F153Aoligonucleotide (CCTTCTAATGGCTTCTCCTTTTCAGCATCAGCGTTATACTTCTTCTTGAA; SEQID NO: 52) anti-sense F151A, F153A oligonucleotide(TTCAAGAAGAAGTATAACGCTGATG CTGAAAAGGAGAAGCCATTAGAAGG; SEQ ID NO:53) wereused for QuikChange site-directed mutagenesis with BnKrp1# 555 as thetemplate. The mutagenesis product was sequenced to verify presence ofdesired mutations and then subcloned into the BamHI/XhoI site ofpET16b-5MYC.

To construct BnKrp1# 598 with multiple amino acids substitution in theCDK binding region (Y149A; F151A; F153A) the sense BnKrp1Y149Aoligonucleotide (AATTTCAAGAAGAAGGCTAACGCTGATGCTGAA; SEQ ID NO:54) andanti-sense BnKrp1Y149A oligonucleotide(TTCAGCATCAGCGTTAGCCTTCTTCTTGAAATT; SEQ ID NO:55) were used forQuikChange site-directed mutagenesis with TopoBnKrp#512 as the template.The mutagenesis product was sequenced to verify presence of desiredmutations and then subcloned into the BamHI/XhoI site of pET16b-5MYC.

To construct BnKrp1# 547, the following two oligonucleotides were usedto amplify the BnKrp1 cDNA lacking the most C-terminal two amino acids,i.e., Ser and Glu that add a 5′ BamHI/NdeI site and XhoI restrictionsite on the 3′ end of BnKrp1 (5′ BnKrp1 BamHI/NdeI:ACGGATCCCATATGGTGAGAAAATGC (SEQ ID NO:26) and 3′ BnKrp1SE>stop XhoI:CTCGAGTCAAGCAGCTAATTTAACCCACTCGTA (SEQ ID NO:56). The PCR fragment wasamplified from pTG #313 (TopoII BnKrp1) and subcloned into the BamHI andXhoI site of pET16b-5myc and sequenced. The resulting vector BnKrp #547contained the BnKrp1 cDNA in frame with the 6×His and myc tags.

To construct BnKrp1# 614, the following two oligonucleotides were usedto amplify the BnKrp1 cDNA lacking the entire CDK binding domain. 5′BnKrp1 BamHI/NdeI: ACGGATCCCATATGGTGAGAAAATGC (SEQ ID NO: 26) and the 3′BnKrp1Δcdk: CTCGAGTCACTTCTTGAAATTATC (SEQ ID NO: 57) which contains aXhoI site. The PCR fragment was amplified from pTG #420 (TopoII BnKrp1)and subcloned into TopoII (Invitrogen) and sequenced. The BamHI/XhoIfragment was then subcloned into pET16b-5myc. The resulting vector BnKrp#614 contained the BnKrp1 cDNA lacking the coding sequence for the CDKbinding region in frame with the 6×His and myc tags.

Example 2 Amino Acid Homology Between Mammalian p27^(KIP1) and PlantKRPs

The CIP1/KIP1 family of CKIs utilizes two contact regions to bind andinhibit the kinase complex. Based on this mode of binding andinhibition, altering the binding capabilities of one of these tworegions could potentially result in a dominant negative protein that canstill interact with the complex (via the intact domain), no longerinhibit the kinase activity and interfere with the wild-type CKI frominhibiting an active cyclin/CDK complex. For example, if the cyclinbinding region were rendered non-functional, the mutant protein wouldstill interact through the CDK binding region with the kinase complexvia the intact domain. Similarly, if the CDK binding region wererendered non-functional, the mutant protein would still interact throughthe cyclin binding region with the kinase complex.

The AtKrp family of CKI's share homology throughout the entire proteinwith the highest homology lying in the most C-terminal 40 to 45 aminoacids (see Wang et al., Nature 386:451-452, 1997; Wang et al, Plant J.15:501-510, 1998; Lui et al., Plant J. 21:379-385, 2000; De Veylder etal., Plant Cell 13:1653-1667, 2001; Jasinski et al., Plant Physiol.130:1871-1882, 2002; Zhou et al., Plant Cell Rep. 20:967-975, 2002; Zhouet al., Plant J. 35:476-489, 2003). However, only the last approximately23 amino acids of the Krp family show homology to the mammalianp27^(Kip1) (see FIG. 1).

In vitro binding experiments were performed to help elucidate thebinding interactions between a representative KRP, namely BnKRP1, andthe AtcyclinD2;1 and the AtCDKA. The binding interactions of other KRPfamily members can also be elucidated using the same in vitro bindingexperiments described below. In vitro binding experiments using mutantversions of BnKrp1 containing amino acid substitutions of highlyconserved amino acids in the CDK binding region revealed that thisregion alone was necessary for the binding to the CDK, while binding tothe Atcyclin was still intact. More importantly an intact CDK bindingdomain is absolutely required for inhibition of the AtCyclinD2/CDKAcomplex. Amino acids lying immediately upstream of the CDK bindingregion, in a region that the present inventors propose is the cyclinbinding region, are conserved among KRP family members (see FIG. 1) butnot in p27^(KIP1). Mutation of several conserved residues within thisproposed cyclin binding domain abrogates the interaction withAtcyclinD2;1, while the interaction with AtCDKA remains intact.Interestingly, this putative cyclin binding region mutant still inhibitsthe AtcyclinD2/CDKA kinase complex yet not as effectively as thewild-type KRP1. The inhibitory concentration that reduces the kinaseactivity by 50% (IC₅₀) was 0.035 μg for the wild-type KRP1 (BnKrp# 461)while the IC₅₀ for the cyclin binding mutant (BnKrp#462) was 1.25 μg.

Similar observations were seen with the mammalian p27^(KIP1) counterpart(see Vlach et al, EMBO J. 16:5334-44, 1997). The IC₅₀ of the p27^(KIP1)cyclin binding mutant was increased in comparison to the wild-typep27^(KIP1). In contrast however, high concentrations of the p27^(KIP1)CDK binding mutant were still capable of inhibiting the kinase complex.This is likely explained by the presence of the 310 helix in mammalianp27^(KIP1) that is absent in the plant Krps (see example 3 for anexplanation).

These data imply that two regions in KRPs exist which are similar to themammalian p27^(KIP1) counterpart responsible for the interaction withthe active kinase complex. Interestingly, the cyclin domain mutant couldstill inhibit the kinase complex, suggesting that the CDK binding regionwas primarily responsible for cyclin/CDK kinase inhibition. Similarlythe cyclin binding region of p27^(KIP1) played an additive role in theinhibition of active CDK complexes. Yet these results illustrate that incontrast to p27^(KIP1), the CDK binding region of KRP1 plays a moresignificant role in kinase inhibition.

Therefore focusing on the high homology between the KRP family andp27^(KIP1) in the CDK binding region residues will be altered toultimately create a dominant negative BnKrp1 molecule that can stillbind the complex, interfere with wild-type BnKRP1 from inhibiting thekinase complex, yet not inhibit the complex itself.

Example 3 Protein Structure of Mammalian p27^(KIP1) in Complex withCyclin/CDK Used to Identify Keys Amino Acids that Contact CDK that areConserved in Plant KRPs

To facilitate the design of the dominant negative Krp molecules thatwould ultimately interfere with the wild-type KRP function thepreviously published structure of mammalian p27^(KIP1) in complex withcyclin A-CDK2 kinase complex was used (see Russo et al., Nature382:325-331, 1996). The structural information along with the alignmentinformation were combined to identify key amino acids that when changedto alanine or other amino acid residues resulted in a protein withdominant negative characteristics. These dominant characteristics are asfollows: 1) mutant Krp bind to cyclin/CDK complexes, 2) mutant proteindo not substantially inhibit the formation of cyclin/CDK complex even athigh concentrations and 3) mutant protein should compete substantiallywith the wild-type KRP molecule for binding to cyclin/CDK complexes. Invivo, mutant Krps that fulfill these characteristics would result inelevated cyclin/CDK kinase activity in the cell that would ultimatelylead to increased cell proliferation and a higher mitotic index.

Mammalian cyclin A/CDK2 kinase activity can be completely shut down bythe binding of p27^(KIP1). The mechanism by which p27^(KIP1) inhibitsthe cyclin A/CDK2 kinase complex has been shown to be a complex process(see Russo et al., supra). p27^(KIP1) utilizes two means to inhibit theactive kinase complex. The first component to the inhibition involvesthe p27^(KIP1) 310 helix that is apparently not conserved in any of theplant KRP family of CKIs. The 310 helix inserts itself into thecatalytic cleft of the kinase to mimic the ATP substrate. Occupation ofthis cleft region by the 310 helix effectively blocks ATP binding andkinase activity. However even in the absence of the 310 helix p27^(KIP1)was still capable of inhibiting the kinase complex (see Polyak et al.,Cell 78:59-66, 1994).

Comparison of the crystal structure of p27^(KIP1) bound to cyclin A/CDK2and the kinase complex alone illustrated that the N-terminal lobe ofCDK2 undergoes significant conformational changes upon bindingp27^(KIP1) (see Russo et al., supra). In fact, specific β-sheets with inthe N-terminal lobe of the kinase that normally help coordinate the ATPin the active site are lost upon p27^(KIP1) binding. Binding ofp27^(KIP1) induces a refolding that involves β-hairpin, β-strandresidues and a 310 helix of p27^(KIP1) and β-sheet amino acids of theN-terminal lobe of CDK2 that refold to form a intermolecular β-sandwich(see id.). This conformation change alone is capable of significantlyinhibiting the kinase activity of cyclin A/CDK2. The residues withinp27^(KIP1) that form this β-sandwich are highly conserved in allmammalian p27^(KIP1) family members and also well-conserved in all KRPfamily members. Based on the results set forth in Example 1, and thelack of a conserved 310 helix, the conserved CDK binding region in theKRP family members likely binds the active kinase complex and inhibitsthe kinase activity by inducing conformational changes in the N-terminallobe of the AtCDKA kinase. However, another region of KRP can not beruled out as actually mimicing ATP binding by inserting into thecatalytic cleft just as the 310 helix does in mammalian p27^(Kip1).

The most carboxy-terminal 23 amino acids of the KRP family show thegreatest amino acid identity to the mammalian p27^(Kip1) (see FIG. 1).Several conserved residues in BnKRP1 were systemically changed based onsequence identity to mammalian p27^(KIP1), play a role in forming theβ-sandwich between the CKI and the kinase complex. Each mutant wascompared to the wild-type BnKRP1 (BnKrp#461) for its ability to inhibitthe kinase complex. In some cases the mutant BnKrp1 was also monitoredfor binding to AtcyclinD2;1 or the AtCDKA. Results for these experimentsare summarized below in Table 2.

Mutations were introduced as described in detail in Example 4, “MutantKRP CKI Polypeptides.”

At the most N-terminal end of the CDK binding region resides the Lys TyrAsn Phe Asp Phe (KYNFDF) motif (SEQ ID NO:58). This motif is highlyconserved in KRP family members from both Arabidopsis and Brassica napusspecies. These residues are for the most part conserved in p27^(KIP1).In p27^(KIP1) they form a portion of a β-hairpin turn that ultimatelyforms the β-sandwich with CDK2 in the cyclin A-CDK2 complex. Each ofthese conserved residues were changed to alanine and tested for theirability to inhibit the kinase complex (see Table 2). Many single aminoacid substitution mutations did not affect the ability to inhibit thekinase complex in vitro. However, BnKrp1 #587 and BnKrp1 #572, showedinteresting results. In each case, kinase inhibition was attenuatedcompared to the wild-type protein suggesting that this region of KRP1plays a role in kinase inhibition.

The side chains of both phenylalanines 62 and 64 in p27^(KIP1) contactCDK2, play an integral role in forming the β-sandwich and are conservedin KRP1. (See FIG. 1.) Since Krp1 F151A (BnKrp1 #572) inhibitoryactivity was partially compromised, both phenylalanines 151 and 153 werechanged to alanine. This double mutant, BnKrp1 #512 no longer inhibitedthe kinase complex despite its ability to still bind the kinase complexvia cyclinD2;1. There may still be some residual binding of this mutantto the CDK portion of the complex.

Tyrosine 149 of KRP1 is not conserved in p27^(KIP1); however, it lies inthe N-terminal position of the β-hairpin that contacts CDK2 and formspart of the β-sandwich. Tyrosine 149 is highly conserved in KRPs familymembers of Arabidopsis and Brassica. When Tyrosine 149 was changed toalanine (BnKrp1 #587), the inhibitory activity was partiallycompromised, suggesting that it plays an important role in the bindingand/or inhibition of CDKA. Therefore, mutation of this position (Y149A)can be combined with the double mutant Krp1 F151A; F153A. This triplemutant was expected to retain its binding to the kinase complex whileloosing its ability to inhibit kinase activity.

The region C-terminal to the KYNFDF motif (SEQ ID NO:58) in KRP1 alsocontains several amino acids that are conserved in p27^(KIP1). (See FIG.1.) Many of these residues are conserved in the β-strand of p27^(KIP1)that forms a portion of the β-sandwich. Several of these conserved aminoacids were changed in pairs to alanine (see Table 2 for summary). In allbut one case, the inhibitory function was not significantly impaired.The exception was the Krp1 #545 (E164A; W165A) mutant that was a muchweaker inhibitor than the wild-type KRP1. The tryptophan 165 extends itsside chain within the β-sandwich. Phenylalanines 151, 153 and E164 andW165 were all changed to alanine (Krp1 #574). This compound mutantBnKrp1 also failed to inhibit the kinase complex even when used at highconcentrations.

Truncation of the complete CDK binding region of KRP1 was also incapableof inhibiting the kinase complex. This was not surprising since theentire region responsible for binding the CDK was deleted. However webelieve it likely that such a truncation will result in an unstableprotein.

Example 4 Mutant KRP CKI Polypeptides

As an improvement over previous techniques used to silence geneexpression at the post-transcriptional RNA level, suppression of KRPfamily members in plants has been accomplished at the protein levelusing a dominant-negative strategy. The basic principle of the dominantnegative strategy is to engineer a gene to produce a mutant protein thatprevents the normal copies of the same protein from performing theirfunction. In this case, the normal KRP protein forms a multi-subunitcomplex with cyclin/CDK complex to inactive the kinase activity.Therefore, expressing a mutant version of wild-type KRP1 will interferewith the normal copies of KRP1 from inhibiting cyclin/CDK kinaseactivity. Furthermore, given the high degree of homology between the 7KRP family members of Brassica, the mutant Krp1 will behave as adominant negative towards other family members or possibly all familymembers. Finally, the cyclin and CDK binding regions of KRPs arewell-conserved in other plant species. Therefore, this dominant negativeKrp1 will protect cyclin/CDK kinase complexes from inhibition byendogenous KRPs in other plants such as, e.g., corn, wheat, Canola, soy,rice, cotton, poplar and the like.

Previously, dominant negative mutants have been used to help elucidatevarious signal transduction pathways. In particular, dominant negativeRas, a GTPase is the most commonly used dominant negative protein todate and has played a major role in the strategy used to study otherGTPases (Feig and Cooper, Mol. Cell. Biol. 8:3235-3243, 1988).Similarly, dominant negative versions of CDKs were used to identifyroles for CDKs in cell cycle control (van den Heuvel and Harlow, Science262:2050-2054, 1993).

The KIP/CIP family of CKIs differs from the INK family of inhibitors inthe mechanism they use to inhibit the kinase complex. While the INKfamily only binds to the CDK, the CIP/KIP family has two conservedregions that independently bind the cyclin and CDK. The fact that theCIP1/KIP1 family of CKIs and the KRP family of CKIs utilize two contactregions to bind the complex makes them an ideal candidate for thedominant negative strategy. This is so because the CDK binding regioncan be targeted for mutants that abolish the interaction with the CDKwhile keeping the region of KRP1 that binds the cyclin intact.

In the present invention, the Canola, Brassica napus (Bn), KRPs andmouse ear cress Arabidopsis thaliana (At) KRP molecules have been clonedand their ability to inhibit cyclin/CDK activity confirmed. To this end,an in vitro assay was developed to test various KRP's ability to inhibitcyclin/CDK complexes. Arabidopsis cyclin D2;1 (AtcyclinD2;1) andArabidopsis CDKA (AtCDKA) were epitope tagged and cloned into abaculovirus expression vector system (BD Biosciences). AtCyclinD2;1 wastagged with the FLAG epitope (Sigma-Aldrich) on the N-terminus. Thehemagglutinin (HA) epitope was placed in frame with the 5′ end ofAtCDKA. Production of AtcyclinD2;1 protein was achieved by infecting S.frugiperda Sf9 cells with AtcyclinD2;1 baculovirus. Production of AtCDKAwas achieved by infection of S. frugiperda Sf9 cells with CDKAbaculovirus. An active complex of cyclin D2;1/CDKA was accomplished byco-infecting of S. frugiperda Sf9 with AtcyclinD2;1 plus AtCDKAbaculovirus. The cyclin, CDK and the active complex were purified andassayed for kinase activity. Kinase activity was monitored using astandard kinase assay with Histone HI (HHI) as the substrate or by usingrecombinant NtRb (Nakagami et al., Plant Cell 14:1847-1857, 2002).Cyclin D2;1 infected insect cells produced no active complex. SimilarlyCDKA infected cells produced no active kinase. When cells were infectedwith both the Atcyclin D2;1 and AtCDKA, kinase activity was easilydetected. Active cyclin CDK complexes can also be purified from plantprotein tissue extracts or from plant tissue culture cell extracts byusing p13suc1 agarose beads (Wang et al., Plant J. 15:501-510, 1998).

Krps were designed to contain a N-terminal poly-histidine epitope tag(HIS tag) by subcloning the Krp cDNA inframe with the coding sequencefor HIS tag in the pET16b vector (Novagen). Wild-type and mutant Krpprotein expression was induced in bacteria and subsequently purified asa HIS tag fusion protein using Ni-agarose.

All wild-type Brassica napus (Bn)Krps tested (BnKRP1, BnKRP4, BnKRP5)were extremely effective at inhibiting the cyclin D2;1/CDKA complex. Inall cases inhibition was also dose responsive. Several other ArabidopsisAtKrps (AtKRP1, AtKRP2) were also effective inhibitors.

CIP1/KIP1 family of CKIs utilize two contact regions to bind and inhibitthe kinase complex. Based on this mode of binding and inhibition,altering the binding capabilities of one of these two regions couldpotentially result in a mutant protein with dominant negativecharacteristics. For example, if the cyclin binding region is renderednon-functional, the mutant protein would still interact through the CDKbinding region with the kinase complex via the intact domain. Similarly,if the CDK binding region is rendered non-functional, the mutant proteinwould still interact through the cyclin binding region with the kinasecomplex.

ICK/KRP family members have limited amino acid identity to the mammalianp27^(KIP1) family. This identity is limited to the most C-terminal 24 to30 amino acids. In fact the location of the cyclin/CDK binding domain ofmammalian p27^(KIP1) is located at the N-terminus of the protein whilethe homologous region in the plant Krps is found at the most C-terminalportion of the protein. (See FIG. 1, showing alignment of p27^(KIP1) andvarious Krp family members.) The cyclin binding region within mammalianp27^(KIP1) is not conserved in plant KRPs. Yet the region immediatelyupstream of the putative CDK binding region is conserved in all plantKRPs. This conserved region although not homologous to p27^(KIP1) couldbe responsible for the interaction with the cyclin. Amino acidsubstitutions of several residues in this proposed cyclin binding regionof BnKRP1 (amino acids 125-138) abolishes binding to the cyclin but doesnot affect binding to the CDK. These data suggest that two regions existin KRPs similar to the mammalian p27^(KIP1) counterpart that mediate theinteraction with the active kinase complex. Interestingly, mutating thecyclin binding region did not completely abolish inhibition of thekinase complex, suggesting that the CDK binding region is primarilyresponsible for cyclin/CDK kinase inhibition.

Given the high degree of homology between the CDK binding regions ofplant and mammalian CKIs, the crystal structure of p27^(KIP1) bound tothe cyclin A/CDK2 complex was used to help identify contact residues ofp27^(KIP1) that bind the cyclin and CDK (Russo et al., Nature382:325-331, 1996). Contact residues were compared to various KRPsequences to determine whether or not they are conserved in the regionthat carries the highest homology to p27^(KIP1). At the most N-terminalend of the CDK binding region lies the LysTyrAsnPheAspPhe (KYNFDF) (SEQID NO:588) motif. These residues are for the most part conserved in p27.They form a α-sheet that contacts CDK2 in the cyclin A-CDK2 complex.

Each of these conserved residues were changed to alanine and tested fortheir ability to inhibit the kinase complex. Interestingly, many ofthese single amino acid substitution mutations did not affect theability to inhibit the kinase complex in vitro. However Krp 1 F151A andKrp1 Y149A mutants did reduce the CKI activity without affecting bindingto the kinase complex.

Since the side chains of both phenylalanines 151 and 153 contact the CDKand Krp1 F151A inhibitory activity was partially compromised, bothphenylalanines 151 and 153 were replaced by alanine. This double mutantBnKrp1 (BnKrp1 F151A; F153A) no longer inhibits the kinase complexdespite its ability to bind the kinase complex via cyclinD2;1. It cannotbe ruled out that there may still be some residual binding of thismutant to the CDK portion of the complex.

Tyrosine 149 in Krp1 is not conserved in p27^(KIP1), however in thestructural model of mammalian p27 a similar amino acid residue lies inthe position of the β-sheet that contacts CDK2. When changed to alanine(Krp1Y149A) the inhibitory activity was partially compromised,suggesting that this amino acid residue plays an important role in thebinding and/or inhibition of CDKA. Therefore, mutation of this position(Y149A) combined with the double mutant Krp1 F151A; F153A produced avariant protein that no longer inhibited the kinase complex. It isexpected that this triple mutant (BnKrp1 Y149A; F151A; F153A) stillretains binding to the kinase complex while unable to inhibit kinaseactivity.

As above, the single amino acid substitutions of Y149A and F151A bothindividually reduced the ability of the mutant BnKrp1 polypeptide frominhibiting the kinase complex. The double mutant of BnKrp1 Y149A; F151Ais expected to produce a mutant protein that no longer inhibits thekinase complex.

The region C-terminal to the KYNFDF motif (SEQ ID NO:58) in KRP1 alsocontains several amino acids that are conserved in p27^(KIP1). Theseconserved amino acids were changed, in pairs to alanine. In all but onecase, the inhibitory function was not significantly impaired. Theexception was Krp1 E164A; W165A mutant that was a much weaker inhibitorthan the wild-type KRP1. In a separate derivative protein,Phenylalanines 151, 153 and E164 and W165 were all replaced by alanine.This multiple mutant of BnKrp1 also failed to inhibit the kinasecomplex.

Truncation of the complete CDK binding region of KRP1 was also incapableof inhibiting the kinase complex. This was not surprising since theentire region responsible for binding the CDK was deleted.

Results of the evaluation of various BnKrp1 mutants for biologicalactivity are summarized in Table 2. TABLE 2 Biological Activity ofBnKrp1 Mutants Construct Cyclin CDK Inhibition of Dominant Number KrpMutation binding binding Kinase Activity Negative?¹ 461 BnKRP1 wild type+++ +++ +++ N/A² 586 BnKrp1 K148A ND³ ND +++ N/A 587 BnKrp1 Y149A ND ND++ − 588 BnKrp1 N150A ND ND +++ N/A 572 BnKrp1 F151A +++ +/− ++ − tbdBnKrp1 D152A ND ND ND N/A 573 BnKrp1 F153A ND ND +++ N/A 512 BnKrp1F151A; F153A +++ − −⁴ ++++ 598 BnKrp1 Y149A; F151A; ND ND − +++++ F153A553 BnKrp1 K157A; P158A ND ND +++ N/A 554 BnKrp1 R162A; Y163A ND ND ++ −555 BnKrp1 E164A; W165A ND ND −⁴ ++ 574 BnKrp1 F151A; F152A; ++ − −⁴ ++E164A; W165A 556 BnKrp1 KL-AA ND ND ND ND 547 BnKrp1 SE-stop ND ND +++N/A¹Protection of kinase complex from inhibition by wild-type BnKrp1.²N/A means not applicable.³ND means not determined.⁴candidate did not inhibit the kinase activity even when used up to 10times the minimum amount of wild-type inhibitor needed to abolish kinaseactivity.

As mentioned previously, disrupting the cyclin binding domain resultedin a mutant Krp1 protein that still retained some inhibitory activity.Nevertheless, there likely exists other amino acids within the putativecyclin binding region (amino acids 125-138) that can be altered tocreate a mutant protein that fulfills the optional dominant negativecharacteristics. In addition, while alanine was used in allsubstitutions in these particular studies, it is expected that replacingthe identified amino acid residues with another non-alanine amino acidresidue that differs significantly in one or more physical properties(other non-conservative substitutions) will also yield a dominantnegative protein. In particular, it is expected that replacing aparticular amino acid residue with an oppositely charged amino acid orwith an amino acid residue with a substantially different definingcharacteristic, such as a hydrophilic residue with a hydrophobid residueand vice versa, and the like, will yield an even better dominantnegative candidate.

The ideal dominant negative candidate will not inhibit the kinaseactivity even when used up to 10 times the minimum amount of wild-typeinhibitor needed to abolish kinase activity. Several mutants fulfilledthis requirement (see Table 2). The importance of this feature lies inthe fact that when expressed in vivo, the levels of the mutant proteinmay be in excess levels compared to the wild-type protein.

The dominant negative BnKrp1 molecules identified in this studyeffectively protect the cyclinD2;1/CDKA complex from wild-type BnKRP1inhibition. The same dominant negative derivative Krp1 molecule alsoprotects the kinase complex from inhibition by KRP molecules from otherspecies such as Maize, Soy, rice, cotton, poplar and alfalfa (seeExample 7).

Example 5 Mutant BnKrp1 Proteins Competitively Block the Wild-Type CKIProtein from Inhibiting the Kinase Complex

Dominant negative candidates that are particularly useful will notinhibit the kinase activity even when used up to 10 times the minimumamount of wild-type inhibitor needed to abolish kinase activity. Severalmutants fulfilled this requirement (see Table 2, FIG. 1, and Example 2).The importance of this feature lies in the fact that when expressed invivo, the levels of the mutant protein may be in excess levels comparedto the wild-type protein, and it is important that the mutant proteindoes not substantially inhibit the kinase complex at virtually anyconcentration.

Several dominant negative candidates were tested for their ability toprotect the cyclinD2;1/CDKA complex from wild-type BnKRP1. Thecompetition experiments were performed as follows. ActivecyclinD2;1/CDKA kinase complex (7 μg) was pre-incubated with candidatedominant negative BnKrp1 mutants (5 μg or 10 μg) for 20 minutes in 1×kinase buffer. The complex was then tested for its kinase activity inthe absence or after the addition of wild-type BnKrp1 (0.5 μg, 1.0 μg or2 μg) using HHI as the substrate. The kinase reactions were thenresolved by SDS PAGE as described in Example 1, “Kinase assay.” Resultswere quantified by phosphorImager (Molecular Dynamics).

BnKrp1 F151A; F153A (BnKrp1 #512) was capable of protecting the kinasecomplex from inhibition by the wild-type BnKRP1. 5 μg of BnKrp1 F151A;F153A restored up to 40% of the kinase activity in the presence of 0.6μg wild-type KRP1. This dominant negative effect was dose dependent, upto 60% of the kinase activity was restored when 10 μg of BnKrp1 F151A;F153A was used.

The mutant BnKrp1 #555 (E164A; W165A) is a promising dominant negativecandidate because it also failed to inhibit the kinase activity evenwhen used at 10 μg per reaction. In competitive experiments, this mutantprotected the active kinase complex from inhibition by wild-type KRP1but only restored activity by 14% when 5 μg was used and by 15% when 10μg was used. The mutations from BnKrp1 #512 and BnKrp1 #555 werecombined into one mutant protein called BnKrp1 #574 (F151A, F153A,E164A, W165A). This compound mutant failed to inhibit the kinaseactivity on its own at 5 μg and 10 μg quantities. This compound mutantwas capable of protecting the kinase complex from inhibition bywild-type BnKRP1 but could only restore activity by as much as 35%. Themutant BnKrp1 # 598 (Y149A; F151A; F153A) was capable of protecting thekinase complex from inhibition by the wild-type BnKRP1. 5 μg of BnKrp1F151A; F153A restored up to 55% of the kinase activity in the presenceof 0.6 μg wild-type KRP1. This dominant negative effect was dosedependent, up to 80% of the kinase activity was restored when 10 μg ofBnKrp1 Y149A; F151A; F153A was used.

These data suggest that the optimal region to target for a dominantnegative is the β-hairpin region. However not just any residue can bealtered. Several single amino acid substitutions failed to create adominant negative Krp (see Example 3) but in fact, changing too many ofthe conserved residues also resulted in a mutant that fulfills most ofthe requirements of the dominant negative except it ultimately failed tobehave as a dominant negative. AtKrp2 is the most closely related toBnKrp1 and AtKrp 1. The mutant AtKrp2 where the KYNFDF motif (SEQ IDNO:58) was changed to KAAAAA (SEQ ID NO:59) had promisingcharacteristics of a dominant negative Krp molecule. At highconcentrations, this BnKrp2 mutant failed to inhibit the kinase complexas expected. Interestingly, this mutant failed to protect the kinasefrom inhibition by wild-type BnKRP1.

In summary: BnKrp1 F151A; F153A blocked the wild-type KRP frominhibiting the kinase, Krp1 E164A; W165A was not as good at blocking thewild-type KRP while BnKrp1 (Y149A; F151A; F153A) was the best dominantnegative candidate in the vitro competition assays. The double mutantBnKrp1(Y149A; F151A) is also likely to be capable of protecting thekinase complex from inhibition.

As mentioned earlier, disrupting the cyclin binding domain resulted in amutant Krp1 protein that retained some inhibitory activity. Neverthelessthere likely exists other amino acids that can be altered to create amutant protein that fulfills all of the dominant negativecharacteristics. Furthermore, alanine was the amino acid of choice forall substitutions. In many of the mutants presented, replacing theresidue with an non-conservative amino acid could yield an even betterdominant negative candidate.

Example 6 BnKrp1 F151A; F153A and BnKrp1 Y149A; F151A; F153A behave as adominant negative towards other Brassica napus KRP family members

One of the overall objectives of the dominant negative strategy was tointroduce a mutation into a single member of the KRP family that behavesas a dominant negative not only towards its wild-type counter part butalso towards all of its family members. The alignment of the KRP familyof CKIs in Brassica napus and in Arabidopsis illustrates the highsequence identity that lies in the extreme C-terminus of the protein.Data has been presented above demonstrating that the conserved regionjust N-terminal to the CDK binding domain is responsible for bindingcyclins. Furthermore, several groups have presented 2-hybrid screeningdata illustrating that several KRP molecules have overlappingspecificity for cyclin binding.

BnKRP4 differs from BnKRP1 in the β-hairpin. Phe 151 is conservedbetween BnKRP1 and BnKRP4, however, BnKRP1 F153 is a proline in BnKRP4.This is not necessarily a conservative amino acid substitution andsuggests that BnKRP4 may interact with the cyclin/CDKA complexdifferently than BnKRP1. However it has already been shown that thisposition within the CDK binding domain does not appear play asignificant role in cyclin/CDKA inhibition (mutant BnKrp1 #573, seeExample 1).

BnKRP4 was cloned using the following two oligonucleotides to amplifythe BnKrp4 cDNA adding a 5′ BamHI/NdeI site and XhoI restriction site onthe 3′ end of BnKrp4-1 (5′ BnKrp4-1 BamHI/NdeI: GGATCCCATATGGGAAAATACATAAAG (SEQ ID NO:60) and 3′ BnKrp4-1 XhoI:CTCGAGCTAATCATCTACCTTCTT (SEQ ID NO:61). The PCR fragment was amplifiedfrom pTG #315 (TopoII BnKrp4-1) and subcloned into the BamHI and XhoIsite of pET16b-5myc and sequenced. The resulting vector BnKrp #605contained the BnKRP4 wild-type cDNA in frame with the 6×His and myctags. The protein was expressed and purified as described in example 1.

BnKrp4 is a potent inhibitor of the AtcyclinD2;1/CDKA kinase complexwith an IC₅₀ very similar to that of wild-type BnKRP1. Competitionexperiments were performed as described in Example 4. In this case,BnKrp1 #512 (F151A; F153A) and BnKrp1 #598 (Y149A; F151A; F153A) wereboth capable of protecting the kinase complex from inhibition bywild-type BnKRP4. Based on this observation, it is expected that BnKrp1#512 and BnKrp1 #598 would behave as a dominant negative towards most,if not all, BnKRP family members.

Example 7 BnKrp1 F151A; F153A and BnKrp1 Y149A; F151A; F153A Behave asDominant Negative Towards Krps of Other Plant Species

The overall objective of the dominant negative strategy was to introduceone or more mutations into a single member of the KRP family that notonly behaves as a dominant negative towards its wild-type family membersbut also against KRP family members across different plant species. Cropplants of interest in the present invention include, but are not limitedto, soybean, canola, corn, wheat, alfalfa, rice, vegetable crops such astomato, and even trees, such as poplar.

The nucleotide sequence of AtKRP1 (GenBank# U94772) was used to performa tBLASTn search for KRP sequences of maize, soy, poplar, tobacco andrice. Several short ESTs showed high homology limited to the cyclin/CDKbinding domains. Several of these short sequences were aligned andillustrate the conservation of this region across various plant species.(See FIG. 2)

In maize, one particular entry, Genbank accession # AY986792 containedan open reading frame of 573 bp, encoding a full length protein withhigh identity to several Bn KRP family members. This protein was calledZmKRP4 but it isn't necessarily the maize homologue of BnKRP4 or AtKRP4.Like BnKRP4, ZmKRP4 differs from BnKRP1 in the α-hairpin. Phe 151 wasconserved between BnKRP1 and BnKRP4 and ZmKRP4. However, BnKRP1 F153 wasa proline in both BnKRP4 and ZmKRP4. ZmKRP4 was amplified from cDNA madefrom total RNA isolated from mature corn tassels. The sequence of theoligonucleotide was gathered from the Genbank accession # AY986792.ZmKRP4 5′BamHI/NdeI: GGATCCCATATGGGCAAGTACATGCGC (SEQ ID NO:62) andZmKRP43′BamHI: GGATCCTCAGTCTAGCTTCACCCA (SEQ ID NO:63). The PCR productwas subcloned into TOPOII (Invitrogen) and sequenced. For proteinproduct of ZmKRP4, the 573 bp BamHI fragment was cloned into the BamHIsite of pET16b-5Myc vector and insert orientation was determined byrestriction enzyme mapping. Recombinant protein was produced asdescribed in Example 4.

Similarly in soy, one particular entry, Genbank accession # AY439104contained an open reading frame of 499 bp, encoding a full lengthprotein with high identity to several Bn KRP family members. Thisprotein called GmKRP2-2 is virtually identical to BnKRP1 within theβ-hairpin. GmKRP2-2 was amplified from cDNA made from total RNA isolatedfrom young developing soy plantlets. Oligonucleotide were designed fromthe sequence of Genbank accession # AY439104: GmKRP2-25′XhoI/NdeI:ctcgaggacatatggagatggctcaggttaaggca (SEQ ID NO:64) and GmKRP2-13′XhoI:ctcgagtcaactgaacccactcgtatcgtcc (SEQ ID NO:65). The PCR product wassubcloned into TOPOII (Invitrogen) and sequenced. For GmKRP2-2 proteinexpression, the 499 bp XhoI fragment was cloned into the XhoI site ofpET16b-5Myc vector and insert orientation was determined by restrictionenzyme mapping. Recombinant protein was produced as described in Example4.

Both ZmKRP4 and Gm Krp2-2 were tested for their ability to inhibitrecombinant cyclinD2;1/CDKA kinase complex. ZmKRP4 was a potentinhibitor of the kinase complex with an IC₅₀ very similar to that ofwild-type BnKRP1, 0.035 μg. Similarly, GmKrp2-2 was also a potentinhibitor of the kinase complex. Both dominant negative BnKrp1 (BnKrp1#512 and #598) were capable of blocking ZmKRP4 and GmKrp2-2 frominhibiting the kinase complex. In each case, the protection wascomparable to the protection that BnKrp1 #512 gave towards BnKRP1inhibition. This result illustrates the cross species effect of thedominant negative Krp of the present invention.

The experiments also suggest that BnKrp #512 and BnKrp #598 will have asimilar effect on other cross-species KRP family members such as thosefrom rice, wheat, sorgum, sugar cane, sugar beets, and the like. KRPmolecules from these plant species, along with others, can be cloned,expressed as recombinant proteins, and evaluated in similar studies todemonstrate that BnKrp1 #512 and BnKrp1 #598 has a similar effect onthese family members.

Example 8 Parallel Mutation in Other Plant Species KRP Molecules Resultsin Dominant Negative Molecules

The residues changed in BnKRP1 to produce the dominant negative effectare conserved in several family members of KRPs in Canola, corn, soy,rice, alfalfa, poplar, tobacco, and the like. The substitution ofconserved phenylalanines to alanine residues can be performed in severalKRPs of these crop plants and tested for their ability to behave asdominant negative molecules.

F151 and F153 are conserved in some but not all KRPs in Canola, soy,corn. Mutation of these conserved residues in soy and corn KRPs willresult in a similar dominant negative Krp molecule.

Example 9 Transgenic Canola Plants Expressing Transgene ConstructsDesigned to Confer Embryo-Specific and Constitutive Expression ofKrp1(F151a; F153a)

Based on the in vitro results present in the above examples, expressionof the invention in cultured plant cells or in transgenic plants ispredicted to increase endogenous CDK kinase activity. This increase inCDK kinase activity will have a positive driving influence on the cellcycle. The invention was cloned into a plant expression vector. A plantexpression vector contains a native or normative promoter linked to theabove described invention. Promoters can range from strong, weak,inducible, tissue-specific, organ specific and developmental specific.

The equivalent of BnKrp1(F151A; F153A) mutations were introduced intoAtKRP1 using the following oligonucleotides: “QC AtKrp1 cds F173A;F175A-coding” 5′-TTCAAGAAGAAGTACAATGCCGATGCCGAGAAGGAGAAGCCATTA-3′ (SEQID NO:66) and “QC AtKrp1 cds F173A; F175A-noncod”5′-TTATGGCTTCTCCTTCTGGGCA TCGGCATTGTACTTCTTCTTGAA-3′ (SEQ ID NO:67).AtKrp1#359 was used as the template using a Stratagene Quikchange SiteDirected Mutagenesis Kit. AtKrp1 F173A; F175A (pTG356) was confirmed bysequencing. The LFAH12 promoter was inserted on the 5′ end of the mutantAtKrp1(F173A; F175A) in pTG 356 in two steps. First, the SalI and PstILFAH12 promoter from pCAMBIA 2381Z (pTG 254) was subcloned intopBluescript II (Stratagene) in the SalI and PstI sites, resulting in pTG357. The LFAH12 promoter was then cut from pTG 357 using PstI and BamHIand inserted into pTG 356 resulting in pTG 361. Finally pTG 361 wasdigested with KpnI and NotI, pLAY112 was digested with NotI and BglII(mas3′ utr component) and pCGN 1547 was digested with KpnI and BamHI anda 3-way ligation was performed. This resulted in a LFAH12-At ICK1 cdsDN-mas 3′ utr cassette (pTG 369).

The plant expression vector containing BnKrp1(F151A; F153A) undercontrol of the constitutive promoter 35S was constructed in thefollowing way. The BamHI/XhoI fragment containing BnKrp1(F151A; F153A)cds was cloned into pTG 271 (35S/TOPO Blunt) using the same sites. Theresulting construct (pTG529) was cut with KpnI and XbaI. pLAY112 was cutwith XbaI and HindIII and pCGN1547 was cut with KpnI and HindIII and a3-way ligation was performed. This resulted in a 35S-BnKrp1(F151A;F153A)-mas3 utr cassette (pTG533).

Canola (Brassica napus) Transformation

The double haploid canola variety DH12075 was transformed with theLFAH-12 AtKrp1(F173A; F175A) and 35-S BnKrp1(F151A; F153A) transgeneexpression constructs (also referred to collectively in this Example as“mutant Krp1 transgene expression constructs”) using anAgrobacterium-mediated transformation method based on that of Maloney etal. (Maloney et al., Plant Cell Reports 8:238, 1989).

Sterilized seeds were germinated on ½ MS (Murashige & Skoog) media with1% sucrose in 15×60 mm Petri dishes for 5 days with approximately 40 toabout 60 seeds per plate. A total of approximately 1500 seeds weregerminated for each transformation construct. Seeds were not fullysubmerged in the germination medium. Germinated seedlings were grown ina tissue culture room at 25° C., on a 16 hour light/8 hour dark cycle.

Cotyledons were cut just above the apical meristem without obtaining anyof the meristem tissue. This was done by gently gripping the twopetioles with forceps immediately above the apical meristem region. Carewas taken not to crush the petioles with the forceps. Using the tips ofthe forceps as a guide, petioles were cut using a scalpel with a sharpNo. 12 blade. Cotyledons were released onto a 15 mm×100 mm plate ofco-cultivation medium. Properly cut cotyledons separate easily. If theydid not, there was a very good chance that meristematic tissue had beenobtained and such cotyledons were not used. Each plate heldapproximately 20 cotyledons. Cotyledon explants were inoculated withAgrobacterium after every few plates that were prepared to avoid wiltingwhich would have a negative impact on following stages of the protocol.

Mutant Krp1 transgene expression constructs were introduced intoAgrobacterium tumefaciens electroporation. Agrobacterium harboring themutant Krp1 transgene expression constructs were grown in AB medium withappropriate antibiotics for two days shaking at 28° C. To inoculatecotyledon explants, a small volume of Agrobacterium culture was added toa 10 mm×35 mm Petri dish. The petiole of each explant was dipped intothe Agrobacterium culture and the cut end placed into co-cultivationmedium in a Petri dish. The plates were sealed and placed in a tissueculture room at 25° C., 16 hour light/8 hour dark for 3 days.

After 3 days, explants were transferred in sets of ten to fresh 25mm×100 mm Petri dishes containing shoot induction medium. This mediumcontained a selection agent (20 mg/L Kanamycin) and hormone (4.5 mg/LBA). Only healthy-looking explants were transferred. Explants were kepton shoot induction medium for 14 to 21 days. At this time, green calliand possibly some shoot development and some non-transformed shoots maycould be observed. Non-transformed shoots were easily recognized bytheir white and purple color. Kanamycin-sensitive shoots were removed bycutting them away and all healthy-looking calli were transferred tofresh plates of shoot induction medium. The explants were kept on theseplates for another 14 to 21 days.

After 2 to 3 weeks, shoots that were dark green in color weretransferred to plates containing shoot elongation medium. This mediumcontained a selection agent (20 mg/L Kanamycin) but did not contain anyhormones. Five shoots were transferred to each plate. The plates weresealed and returned to the tissue culture room. Transformed shoots thatappeared vitrious were transferred to shoot elongation medium containingphloroglucinol (150 mg/L). Shoots that became healthy and green werereturned to shoot elongation medium plates. Repeated transfers ofvitrious shoots to fresh plates of the same medium were required in somecases to obtain normal looking shoots.

Shoots with normal morphology were transferred to 4 oz. baby food jarswith rooting medium containing 0.5 mg/L indole butyric acid. Any excesscallus was cut away when transferring shoots to the jars. Shoots couldbe maintained in jars indefinitely by transferring them to fresh jarscontaining 0.2 mg/L indole butyric acid approximately every 6 weeks.

Once a good root system had formed, the T₀ generation shoots wereremoved from jars, agar removed from the roots, and the plantlettransferred to potting soil. Each independent T₀ plantlet represents anindependent occurrence of insertion of the transgene into the canolagenome and is referred to as an event. A transparent cup was placed overthe plantlet for a few days, allowing the plant to acclimatize to thenew environment. Once the plant had hardened, the cup was removed. TheT₀ transgenic events were then grown to maturity in the greenhouse andT₁ seeds collected.

T₀ Event Characterization

The number of transgene insertion site loci was determined in each eventby Southern analysis. Mutant Krp1 transgene expression in the T₀ eventswas verified by Northern analysis or end-point RT-PCR. Mutant Krp1transgene expression data were obtained for a single time point inembryo development, 19 days after pollination (DAP). From these data itwas concluded that, at this developmental time point, the LFAH12promoter was driving high levels of AtKrp1(F173A; F175A) mRNA. The 35SBnKrp1 (F151A; F153A) transgene expression was monitored in leaf sampleswhich had moderate to high level expression in many events. Theexpression data demonstrated that both promoters were functional indriving mutant Krp1 transgene expression in transgenic canola plants.

Kinase activity can also be measured to determine effect of mutant Krp1protein expression on endogenous Cyclin-CDK kinase activity. Equivalentamounts of protein extracts from transgenic and non-transgenic planttissue or cells are enriched for CDK using p13Suc1 resin (Upstate,Chicago, Ill.) and Histone kinase assays are performed as described inexample 4 or by Wang et al., Plant J. 15:501-510, 1998. Kinase reactionsare resolved by SDS-PAGE and HHI phosphorylation quantified byPhosphorImager.

T₀ plants were successfully generated for both mutant Krp1 transgeneexpression constructs. The tested constructs included (a)LFAH12/AtKrp1(F173A; F175A); (b) 35S BnKrp1(F151A; F153A).

Example 10 Expression In Vivo Accelerates Growth/Early Germination

Expression of the mutant proteins of the present invention in transgenicplants will exert their effects by elevating cyclin-CDK kinase activity.This will have a positive driving influence on the cell cycle thatultimately results in increased organ size. In plants, reducing Krp1expression levels by suppressing expression of an endogenous Krp gene inthe embryo using an inverted repeat approach results in increased seedsize and crop yield. In addition to an effect on yield, increased seedsize can result in increased vigor or the seed. Larger seeds contain agreater amount of stored protein and starch reserves to be utilizedduring germination and seedling growth. This can result in earliergermination and more rapid early growth. Early germination and earlyrapid growth are agronomically valuable traits since they lead to morerapid establishment of the crop. This increases the chances of asuccessful crop by reducing the window of vulnerability to harshenvironmental conditions and the like that can damage or ruin a cropbefore establishment.

Increased growth and developmental pace were observed ingreenhouse-germinated seedlings of transgenic canola plants transformedwith the AtKrp1(F173A; F175A) dominant negative expression construct(pTG369) described in Example 9. Twenty-four seeds from each of fifteenindependent transformation events of the AtKrp1(F173A; F175A) dominantnegative expression construct were planted in Jiffy peat pellets intrays and germinated in a greenhouse. Twenty four seeds from each offifteen independent events transformed with the wild type KRP1 gene,also under control of the LFAH 12 promoter, were planted in parallel.Additionally, the untransformed parental DH12075 canola variety and atotal of 135 transgenic events transformed with any of nine unrelatedtransgene constructs were planted at the same time.

After three weeks growth, all seedlings were transplanted to a field tobe grown to seed. Each set of twenty four seedlings for each event wasplanted as an individual plot. At transplantation, there were readilyobserved differences in growth among the seedlings between plots. Acrossthe entire seedling population for all transgene constructs, size variedfrom 1 inch tall to about 4 to 5 inches tall. Developmental progressalso varied, ranging from a few seedlings having only cotyledons presentto seedlings with several true leaves. The untransformed parentalvariety plots fell in the middle of this range. Differences in thetransgenic plots were construct specific. Most of the plots in the fieldthat contained seedlings at the top end of the size and developmentrange were plots containing plants transformed with the AtKrp1(F173A;F175A) dominant negative expression construct (pTG369). Other constructsdid not show this characteristic with most plots showing growth anddevelopment comparable to or less than the untransformed parentalvariety. These results indicate that expression of a dominant negativemutant Krp transgene confers an increased early growth rate andaccelerated developmental pace.

Further characteristics to be monitored include, for example,pre-emergence/germination duration, time of emergence of cotyledon, sizeof cotyledon, time of first true leaf exposed, and second true leafexposure. Rosette diameter can also be monitored in transgenic andnon-transgenic plants. Other characteristics can also be monitoredbetween the transgenic and non-transgenic plants and include, forexample, time of bud emergence and size of the bud bolt, final size ofmain raceme, time of the first flower to emerge, timing of first podappearance, and the like.

Example 11 Expression In Vivo Gives Better Rooting

Canola seeds are very small, require relatively high moisture forgermination and must be planted close to the soil surface to maximizeseedling vigor. Small seeds and shallow planting make the cropvulnerable to abiotic stresses such as dry soil, and flooding.Ubiquitous expression or expression early in root development of themutant polypeptides of the present invention under control of variouspromoters would give accelerated root growth and development. Increasedcell division during root development will benefit seedling vigor byestablishing a firm root base sooner and possibly a larger root basethan non-transgenic plant control. The size of the transgenic plantsroot system can be compared to non-transgenic plant.

Example 12 Evaluation of the Effect of AtKrp1(F173A; F175A) TransgeneExpression During Embryo Development on Canola Yield in Replicated FieldTrials

In this example the transgenic canola plants comprising the ArabidopsisKrp1(F173A; F175A) transgene under the control of the LFAH12 embryospecific promoter (pTG369) was tested in field trials.

Advancement of Transgenic AtKrp1(F173A; F175A) Events to Field Trials.

T₀ events were selected for advancement to field trials based on acombination of transgene expression and transgene insertion locusnumber. Events with verified transgene expression and a single transgeneinsertion locus were assigned the highest priority to be carried forwardto field testing. In some instances, events with multiple insertion lociwere selected if the presence of multiple genes gave a high overalltransgene expression level due to gene dosage.

T₁ seeds from selected events were grown as segregating T₁ populationsin field plots. Each event was planted as a two row, twenty four plantplot. For events with a single transgene insertion locus, segregation ofthe transgene among the twenty four T₁ plants would produce adistribution of approximately six null plants lacking the transgene,twelve heterozygous plants, and six homozygous plants. Each T₁ plant wasindividually bagged before flowering to prevent out-crossing. T₂ seedsfrom each of the twenty four T₁ plants were harvested separately.

The T₂ seed stocks were used to identify which of the twenty four parentT₁ plants were null, heterozygous, or homozygous. Approximately thirtyT₂ seeds from each T₁ plant were germinated on filter paper in petridishes with a solution containing the antibiotic G418, an analog ofkanamycin. Since the plants were co-transformed with the nptIIresistance gene as a selectable marker, only those seeds carrying thetransgene would germinate and continue to grow. If all the seeds on aplate proved to be sensitive to G418, then the T₁ parent was identifiedas a null line. If all the seeds on a plate were resistant to G418, thenthe T₁ parent was identified as a homozygous line. If approximately onequarter of the seeds on a plate were sensitive and the rest resistant,the T₁ parent was identified as a heterozygous line. T₂ seeds fromhomozygous T₁ parents from the same transformation event were bulked togenerate homozygous seed stocks for field trial testing. T₂ seeds fromnull T₁ parents from the same transformation event were bulked togenerate null sibling seed stocks for field trial testing.

Field Trial Design

The effect of the AtKrp1(F173A; F175A) transgene on yield traits in thetransgenic canola lines was evaluated by comparing each transgenic linedirectly with its null sibling in the field in large scale replicatedtrials. Since the null sibling arises from segregation of the transgenein the T₁ generation, the null and homozygous siblings are nearlyidentical genetically. The only significant difference is the presenceor absence of the AtKrp1(F173A; F175A) transgene. This near geneticidentity makes the null sibling the optimal control for evaluation ofthe effect of the AtKrp1(F173A; F175A) transgene. As the main objectiveof the trial was the comparison of the transgenic line from an event toits null segregate, a split plot design was chosen. This design gives ahigh level of evaluation to the interaction between the transgenic andnon-transgenic subentries and the differences between transgenicsubplots between events (the interaction of subplot and main plot) and alower level of evaluation to the differences between overall events orthe main plot.

Field trials were conducted at multiple locations across the prairieprovinces to assess yield phenotypes under the range of environmentalconditions in which canola is typically grown. At all locations, eachtransgenic event was physically paired with its null sibling in adjacentplots. Each plot pair of homozygous and null siblings was replicatedfour times at each trial location. The locations of the four replicateplot pairs in each trial were randomly distributed at each triallocation. Plots were 1.6 m by 6 m and planted at a density ofapproximately 142 seeds per square meter. Plants were grown to maturityusing standard agronomic practices typical to commercial production ofcanola.

Example 13 Increased Yield in Transgenic Canola Expressing AtKrp1(F173A;F175A) During Embryo Development Using Embryo-Specific Promoters

All plots at each yield field trial location were individually harvestedwith a combine. Total seed yield data were collected as total seedweight adjusted for moisture content from each plot. For everytransgenic event in each trial, the mean of the total yield from thefour replicate plots of each homozygous line was compared to the mean ofthe total yield from the four replicate plots of the associated nullsibling line. This comparison was used to evaluate the effect of theAtKrp1(F173A; F175A) transgene on total seed yield. Results from each ofthe multiple trial locations were combined to give an across trialsanalysis of the effect of the AtKrp1(F173A; F175A) transgene on totalseed yield. Statistical analysis of variance at each trial locationpermitted the assignment of a threshold for significance (P=0.05) fordifferences in total seed yield between homozygous transgenic lines andtheir null siblings.

The transgenic AtKrp1(F173A; F175A) canola lines that showed astatistically significant increase in total seed yield are summarized inTable 3. These results demonstrate that over expression of AtKrp 1(F173A; F175A) using an embryo-specific promoter (LFAH12) results inincreased seed yield. TABLE 3 Change in total seed yield in homozygousAtKrp1(F173A; F175A) plants relative to their null siblings. All valuesare statistically significant (P = 0.05) % Yield Event PromoterTransgene Increase TG39-2 LFAH12 AtKrp1(F173A; F175A) 13.0 TG39-26LFAH12 AtKrp1(F173A; F175A) 18.6 TG39-32 LFAH12 AtKrp1(F173A; F175A)12.0

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentireties for all purposes.

1. An isolated, mutant plant CKI polypeptide comprising: a CKI aminoacid sequence having at least one modification relative to a wild-typeplant CKI polypeptide, said wild-type CKI polypeptide comprising (a) acyclin binding region conferring binding affinity for a cyclin and (b) aCDK binding region conferring binding affinity for a CDK; wherein saidat least one modification is within the CDK binding region so as toconfer, relative to the wild-type CKI polypeptide, a modified bindingaffinity of the mutant CKI polypeptide for the CDK; wherein the mutantCKI polypeptide substantially maintains, relative to the wild-type CKIpolypeptide, binding affinity for the cyclin; and wherein the mutant CKIpolypeptide can compete with said wild-type CKI for binding to the CDKbinding region.
 2. The mutant plant CKI polypeptide of claim 1, whereinsaid wild-type plant CKI polypeptide is a KIP related protein (KRP)family member.
 3. The mutant plant CKI polypeptide of claim 2, whereinsaid KRP family member is a Brassica napus, Arabidopsis thaliana,Glycine max, maize, wheat, rice, alfalfa, cotton, or poplar CKIpolypeptide.
 4. The mutant plant CKI polypeptide of claim 2, whereinsaid KRP family member is an Arabidopsis or a Brassica KRP1 polypeptide.5. The mutant plant CKI polypeptide of claim 2, wherein said at leastone modification is within a region corresponding to amino acids 145-168of Brassica KRP1 (BnKRP1).
 6. The mutant plant CKI polypeptide of claim5, which comprises at least two modifications within the regioncorresponding to amino acids 145-168 of Brassica KRP1 (BnKRP1).
 7. Themutant plant CKI polypeptide of claim 5, wherein said at least onemodification comprises an amino acid substitution.
 8. The mutant plantCKI polypeptide of claim 7, wherein the amino acid substitution is anon-alanine to alanine substitution or a non-conservative substitution.9. The mutant plant CKI polypeptide of claim 7, which comprises at leasttwo amino acid substitutions, each amino acid substitution being at aposition independently selected from the group consisting of: (a) aposition corresponding to amino acid 145 of BnKRP1; (b) a positioncorresponding to amino acid 149 of BnKRP1; (c) a position correspondingto amino acid 151 of BnKRP1; (d) a position corresponding to amino acid153 of BnKRP1; (e) a position corresponding to amino acid 164 of BnKRP1;and (f) a position corresponding to amino acid 165 of BnKRP1.
 10. Themutant plant CKI polypeptide of claim 9, wherein one or more, andoptionally all, of the at least two amino acid substitutions arenon-alanine to alanine substitution(s) or non-conservativesubstitutions(s).
 11. The mutant plant CKI polypeptide of claim 5,wherein said at least one modification is within the KYNFDF motif. 12.The mutant plant CKI polypeptide of claim 11, which comprises at leasttwo modifications within the KYNFDF motif.
 13. The mutant plant CKIpolypeptide of claim 12, wherein said at least two modifications withinthe KYNFDF motif comprise amino acid substitutions at positionscorresponding to amino acids 151 and 153 of BnKRP1.
 14. The mutant plantCKI polypeptide of claim 13, wherein at least one of the amino acidsubstitutions at the positions corresponding to amino acids 151 and 153of BnKRP1 is a non-alanine to alanine substitution or a substitution ofan amino acid with an oppositely charged amino acid.
 15. The mutantplant CKI polypeptide of claim 14, wherein each of the amino acidsubstitutions at the positions corresponding to amino acids 151 and 153of BnKRP1 is a non-alanine to alanine substitution or a substitution ofan amino acid with an oppositely charged amino acid.
 16. The mutantplant CKI polypeptide of claim 13, further comprising an amino acidsubstitution at a position corresponding to amino acid 149 of BnKRP1.17. The mutant plant CKI polypeptide of claim 16, wherein the amino acidsubstitution at the position corresponding to amino acid 149 of BnKRP1is a non-alanine to alanine substitution or a non-conservativesubstitution.
 18. The mutant plant CKI polypeptide of claim 13 or 16,further comprising an amino acid substitution at least one of (a) aposition corresponding to amino acid 164 of BnKRP1, and (b) a positioncorresponding to amino acid 165 of BnKRP1.
 19. The mutant plant CKIpolypeptide of claim 18, wherein the amino acid substitution at leastone of (a) and (b) is a non-alanine to alanine substitution or anon-conservative substitution.
 20. The mutant plant CKI polypeptide ofclaim 18, which comprises an amino acid substitution at each of (a) and(b).
 21. The mutant plant CKI polypeptide of claim 20, wherein each ofthe amino acid substitutions at (a) and (b) is a non-alanine to alaninesubstitution or a non-conservative substitution.
 22. The mutant plantCKI polypeptide of claim 5, which is a mutant BnKRP1 polypeptide. 23.The mutant BnKRP1 polypeptide of claim 22, which is (1) BnKRP1 F151A;F153A; (2) BnKRP1 Y149A; F151A; F153A; (3) BnKRP1 E164A; W165A; or (4)BnKRP1 F151A; F153A; E164A; W165A.
 24. The mutant CKI polypeptide ofclaim 1, wherein said at least one modification comprises truncation ofthe CDK binding region.
 25. The mutant CKI polypeptide of claim 24,which is a mutant BnKRP1 polypeptide.
 26. A recombinant nucleic acidencoding the mutant CKI polypeptide of claim
 1. 27. A vector comprisinga replicon and the recombinant nucleic acid of claim
 26. 28. The vectorof claim 27, which is an expression vector further comprising a promoterregion operably linked to the recombinant nucleic acid.
 29. Theexpression vector of claim 28, wherein the promoter region is operablein a plant cell.
 30. The expression vector of claim 29, wherein thepromoter region comprises a CaMV 35S promoter or a LFAH12 promoter. 31.The expression vector of claim 28, wherein the promoter region istranscriptionally active in a tissue- and/or organ-specific fashion. 32.A host cell comprising the recombinant nucleic acid of claim
 26. 33. Ahost cell comprising the vector of claim
 27. 34. A method of producing amutant CKI polypeptide, said method comprising: culturing the host cellof claim 32 or 33 under conditions suitable for expression of thenucleic acid encoding the mutant CKI polypeptide.
 35. The method ofclaim 34, further comprising recovering said mutant CKI polypeptide. 36.A transgenic plant comprising a transgene encoding a mutant CKIpolypeptide, said transgenic plant expressing a wild-type CKIpolypeptide, said wild-type CKI polypeptide comprising (a) a cyclinbinding region conferring binding affinity for a cyclin and (b) a CDKbinding region conferring binding affinity for a CDK, wherein saidmutant CKI polypeptide comprises a CKI amino acid sequence having atleast one modification relative to a reference CKI polypeptide, saidreference CKI polypeptide selected from the group consisting of (1) thewild-type plant CKI polypeptide expressed by the transgenic plant, and(2) a wild-type CKI polypeptide heterologous to (1) and capable ofproviding wild-type CKI function within a cell of the transgenic plantsubstantially equivalent to the wild-type CKI function of (1); whereinsaid at least one modification is within the CDK binding region so as toconfer, relative to the reference CKI polypeptide, a modified bindingaffinity of the mutant CKI polypeptide for the CDK, and wherein themutant CKI polypeptide substantially maintains, relative to thereference CKI polypeptide, binding affinity for the cyclin.
 37. Thetransgenic plant of claim 36, which is a monocotyledonous plant.
 38. Thetransgenic plant of claim 36, which is a dicotyledonous plant.
 39. Thetransgenic plant of claim 36, wherein said reference CKI polypeptide isa KIP related protein (KRP) family member.
 40. The transgenic plant ofclaim 39, wherein said KRP family member is a Brassica napus,Arabidopsis thaliana, Glycine max, maize, wheat, rice, alfalfa, cotton,or poplar CKI polypeptide.
 41. The transgenic plant of claim 39, whereinsaid KRP family member is a KRP1 polypeptide.
 42. The transgenic plantof claim 39, wherein said at least one modification is within a regioncorresponding to amino acids 145-168 of Brassica KRP1 (BnKRP1).
 43. Thetransgenic plant of claim 42, wherein said mutant CKI polypeptidecomprises at least two modifications within a region corresponding toamino acids 145-168 of Brassica KRP1 (BnKRP1).
 44. The transgenic plantof claim 42, wherein said at least one modification comprises an aminoacid substitution.
 45. The transgenic plant of claim 44, wherein theamino acid substitution is a non-alanine to alanine substitution or asubstitution of an amino acid with an oppositely charged amino acid. 46.The transgenic plant of claim 42, wherein said at least one modificationis within the KYNFDF motif.
 47. The transgenic plant of claim 46,wherein said mutant CKI polypeptide comprises at least two modificationswithin the KYNFDF motif.
 48. The transgenic plant of claim 46, whereinsaid at least two modifications within the KYNFDF motif comprise aminoacid substitutions at positions corresponding to amino acids 151 and 153of BnKRP1.
 49. The transgenic plant of claim 48, wherein at least one ofthe amino acid substitutions at the positions corresponding to aminoacids 151 and 153 of BnKRP1 is a non-alanine to alanine substitution ora substitution of an amino acid with an oppositely charged amino acid.50. The transgenic plant of claim 49, wherein each of the amino acidsubstitutions at the positions corresponding to amino acids 151 and 153of BnKRP1 is a non-alanine to alanine substitution or a substitution ofan amino acid with an oppositely charged amino acid.
 51. The transgenicplant of claim 48, wherein the mutant CKI polypeptide further comprisesan amino acid substitution at a position corresponding to amino acid 149of BnKRP1.
 52. The transgenic plant of claim 51, wherein the amino acidsubstitution at the position corresponding to amino acid 149 of BnKRP1is a non-alanine to alanine substitution or a non-conservativesubstitution.
 53. The transgenic plant of claim 48 or 51, wherein themutant CKI polypeptide further comprises an amino acid substitution atleast one of (a) a position corresponding to amino acid 164 of BnKRP1,and (b) a position corresponding to amino acid 165 of BnKRP1.
 54. Thetransgenic plant of claim 53, wherein the amino acid substitution atleast one of (a) and (b) is a non-alanine to alanine substitution or anon-conservative substitution.
 55. The transgenic plant of claim 53,wherein the mutant CKI polypeptide comprises an amino acid substitutionat each of (a) and (b).
 56. The transgenic plant of claim 55, whereineach of the amino acid substitutions at (a) and (b) is a non-alanine toalanine substitution or a non-conservative substitution.
 57. Thetransgenic plant of claim 39, wherein the reference CKI polypeptide is aBnKRP1 polypeptide.
 58. The transgenic plant of claim 57, wherein themutant CKI polypeptide is (1) BnKRP1 F151A; F153A; (2) BnKRP1 Y149A;F151A; F153A; (3) BnKRP1 E164A; W165A; or (4) BnKRP1 F151A; F153A;E164A; W165A.
 59. The transgenic plant of claim 36, wherein said atleast one modification comprises truncation of the CDK binding region.60. The transgenic plant of claim 59, wherein the reference CKIpolypeptide is a BnKRP1 polypeptide.
 61. The transgenic plant of claim36, which is selected from the group consisting of Brassica napus,Arabidopsis thaliana, Glycine max, maize, wheat, rice, alfalfa, cotton,poplar, and camelina.
 62. The transgenic plant of claim 61, which isselected from the group consisting of Brassica napus, Glycine max, andMaize.
 63. The transgenic plant of claim 62, wherein the reference CKIpolypeptide is an Arabidopsis thaliana CKI polypeptide.
 64. A method ofproducing the transgenic plant of claim 36, comprising: introducing intoa plant a vector comprising the transgene encoding the mutant CKIpolypeptide.
 65. A method of modulating cell division in a plant cellexpressing a wild-type CKI polypeptide, said wild-type CKI polypeptidecomprising (a) a cyclin binding region conferring binding affinity for acyclin and (b) a CDK binding region conferring binding affinity for aCDK, the method comprising: expressing within the cell a mutant CKIpolypeptide comprising a CKI amino acid sequence having at least onemodification relative to a reference CKI polypeptide, said reference CKIpolypeptide selected from the group consisting of (1) the wild-typeplant CKI polypeptide expressed within the plant cell, and (2) awild-type CKI polypeptide heterologous to (1) and capable of providingwild-type CKI function within the plant cell substantially equivalent tothe wild-type CKI function of (1); wherein said at least onemodification is within the CDK binding region so as to confer, relativeto the reference CKI polypeptide, a modified binding affinity of themutant CKI polypeptide for the CDK, and wherein the mutant CKIpolypeptide substantially maintains, relative to the reference CKIpolypeptide, binding affinity for the cyclin; and allowing said mutantCKI polypeptide to inhibit wild-type CKI biological activity within theplant cell.
 66. The method of claim 65, wherein said reference CKIpolypeptide is a KIP related protein (KRP) family member.
 67. The methodof claim 66, wherein the mutant CKI polypeptide antagonizes a pluralityof endogenous KRP family members within the plant cell.
 68. The methodof claim 66, wherein said KRP family member is a Brassica napus,Arabidopsis thaliana, Glycine max, or Maize CKI polypeptide.
 69. Themethod of claim 66, wherein said KRP family member is a KRP1polypeptide.
 70. The method of claim 66, wherein said at least onemodification is within a region corresponding to amino acids 145-168 ofBrassica KRP1 (BnKRP1).
 71. The method of claim 70, wherein the mutantCKI polypeptide comprises at least two modifications within a regioncorresponding to amino acids 145-168 of Brassica KRP1 (BnKRP1)
 72. Themethod of claim 70, wherein said at least one modification comprises anamino acid substitution.
 73. The method of claim 72, wherein the aminoacid substitution is a non-alanine to alanine substitution or asubstitution of an amino acid with an oppositely charged amino acid. 74.The method of claim 70, wherein said at least one modification is withinthe KYNFDF motif.
 75. The method of claim 74, wherein the mutant CKIpolypeptide comprises at least two modifications within the KYNFDFmotif.
 76. The method of claim 75, wherein said at least twomodifications within the KYNFDF motif comprise amino acid substitutionsat positions corresponding to amino acids 151 and 153 of BnKRP1.
 77. Themethod of claim 76, wherein at least one of the amino acid substitutionsat the positions corresponding to amino acids 151 and 153 of BnKRP1 is anon-alanine to alanine substitution or a substitution of an amino acidwith an oppositely charged amino acid.
 78. The method of claim 77,wherein each of the amino acid substitutions at the positionscorresponding to amino acids 151 and 153 of BnKRP1 is a non-alanine toalanine substitution or a substitution of an amino acid with anoppositely charged amino acid.
 79. The method of claim 76, wherein themutant CKI polypeptide further comprises an amino acid substitution at aposition corresponding to amino acid 149 of BnKRP1.
 80. The method ofclaim 79, wherein the amino acid substitution at the positioncorresponding to amino acid 149 of BnKRP1 is a non-alanine to alaninesubstitution or a non-conservative substitution.
 81. The method of claim76 or 79, wherein the mutant CKI polypeptide further comprising an aminoacid substitution at least one of (a) a position corresponding to aminoacid 164 of BnKRP1, and (b) a position corresponding to amino acid 165of BnKRP1.
 82. The method of claim 81, wherein the amino acidsubstitution at least one of (a) and (b) is a non-alanine to alaninesubstitution or a non-conservative substitution.
 83. The method of claim81, wherein the mutant CKI polypeptide comprises an amino acidsubstitution at each of (a) and (b).
 84. The method of claim 83, whereineach of the amino acid substitutions at (a) and (b) is a non-alanine toalanine substitution or a non-conservative substitution.
 85. The methodof claim 69, wherein the reference CKI polypeptide is a BnKRP1polypeptide.
 86. The method of claim 85, wherein the mutant CKIpolypeptide is selected from the group consisting of (1) BnKRP1 F151A;F153A; (2) BnKRP1 Y149A; F151A/F153A; (3) BnKRP1 E164A; W165A; or (4)BnKRP1 F151A; F153A; E164A; W165A.
 87. The method of claim 65, whereinsaid at least one modification comprises truncation of the CDK bindingregion.
 88. The method of claim 87, wherein the reference CKIpolypeptide is a BnKRP1 polypeptide.
 89. The method of claim 65, furthercomprising introducing into the plant cell a recombinant nucleic acidencoding the mutant CKI polypeptide.
 90. The method of claim 89, whereinthe recombinant nucleic acid is an expression vector.
 91. The method ofclaim 65, wherein the plant cell is from a plant selected from the groupconsisting of Brassica napus, Arabidopsis thaliana, Glycine max, andMaize.
 92. The method of claim 91, wherein the plant is selected fromthe group consisting of Brassica napus, Glycine max, and Maize.
 93. Themethod of claim 92, wherein the reference CKI polypeptide is anArabidopsis thaliana CKI polypeptide.
 94. A method for increasing plantvigor, comprising: expressing within the plant a mutant KRP polypeptidecomprising a KRP amino acid sequence having at least one modificationrelative to a reference KRP polypeptide, said reference KRP polypeptideselected from the group consisting of (1) the wild-type KRP polypeptideexpressed within the plant, and (2) a wild-type KRP polypeptideheterologous to (1) and capable of providing wild-type KRP functionwithin a cell of the plant substantially equivalent to the wild-type KRPfunction of (1); wherein said at least one modification is within theCDK binding region so as to confer, relative to the reference KRPpolypeptide, a modified binding affinity of the mutant KRP polypeptidefor the CDK, and wherein the mutant KRP polypeptide substantiallymaintains, relative to the reference KRP polypeptide, binding affinityfor the cyclin; and allowing said mutant KRP polypeptide to inhibitwild-type KRP biological activity within the plant so as to increaseplant vigor.
 95. A method for increasing root mass in a plant,comprising: expressing within the plant a mutant KRP polypeptidecomprising a KRP amino acid sequence having at least one modificationrelative to a reference KRP polypeptide, said reference KRP polypeptideselected from the group consisting of (1) the wild-type KRP polypeptideexpressed within the plant, and (2) a wild-type KRP polypeptideheterologous to (1) and capable of providing wild-type KRP functionwithin a cell of the plant substantially equivalent to the wild-type KRPfunction of (1); wherein said at least one modification is within theCDK binding region so as to confer, relative to the reference KRPpolypeptide, a modified binding affinity of the mutant KRP polypeptidefor the CDK, and wherein the mutant KRP polypeptide substantiallymaintains, relative to the reference KRP polypeptide, binding affinityfor the cyclin; and allowing said mutant KRP polypeptide to inhibitwild-type KRP biological activity within the plant so as to increaseroot mass in the plant.
 96. A method for increasing plant seed size,comprising: expressing within the plant a mutant KRP polypeptidecomprising a KRP amino acid sequence having at least one modificationrelative to a reference KRP polypeptide, said reference KRP polypeptideselected from the group consisting of (1) the wild-type KRP polypeptideexpressed within the plant, and (2) a wild-type KRP polypeptideheterologous to (1) and capable of providing wild-type KRP functionwithin a cell of the plant substantially equivalent to the wild-type KRPfunction of (1); wherein said at least one modification is within theCDK binding region so as to confer, relative to the reference KRPpolypeptide, a modified binding affinity of the mutant KRP polypeptidefor the CDK, and wherein the mutant KRP polypeptide substantiallymaintains, relative to the reference KRP polypeptide, binding affinityfor the cyclin; and allowing said mutant KRP polypeptide to inhibitwild-type KRP biological activity within the plant so as to increaseplant seed size.
 97. A method for increasing early germination in aplant, comprising: expressing within the plant a mutant KRP polypeptidecomprising a KRP amino acid sequence having at least one modificationrelative to a reference KRP polypeptide, said reference KRP polypeptideselected from the group consisting of (1) the wild-type KRP polypeptideexpressed within the plant, and (2) a wild-type KRP polypeptideheterologous to (1) and capable of providing wild-type KRP functionwithin a cell of the plant substantially equivalent to the wild-type KRPfunction of (1); wherein said at least one modification is within theCDK binding region so as to confer, relative to the reference KRPpolypeptide, a modified binding affinity of the mutant KRP polypeptidefor the CDK, and wherein the mutant KRP polypeptide substantiallymaintains, relative to the reference KRP polypeptide, binding affinityfor the cyclin; and allowing said mutant KRP polypeptide to inhibitwild-type KRP biological activity within the plant so as to increaseearly germination in the plant.